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Endogenous glucocorticoid analysis by liquid chromatography–tandem mass spectrometry in routine clinical laboratories
Accepted Manuscript Title: Endogenous glucocorticoid analysis by liquid chromatography-tandem mass spectrometry in routine clinical laboratories Author: James M. Hawley Brian G. Keevil PII: DOI: Reference:
S0960-0760(16)30144-3 http://dx.doi.org/doi:10.1016/j.jsbmb.2016.05.014 SBMB 4729
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Revised date: Accepted date:
2-9-2015 11-5-2016 12-5-2016
Please cite this article as: James M.Hawley, Brian G.Keevil, Endogenous glucocorticoid analysis by liquid chromatography-tandem mass spectrometry in routine clinical laboratories, Journal of Steroid Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2016.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Endogenous glucocorticoid analysis by liquid chromatography-tandem mass spectrometry in routine clinical laboratories.
James M Hawley 1 and Brian G Keevil 1,2
1. University Hospital South Manchester, Manchester, UK. 2. Manchester Healthcare Academy, Manchester, UK
Corresponding author: James M Hawley Address: Clinical Biochemistry Department, Clinical Sciences Building, University Hospital South Manchester, Southmoor Road, Manchester, M23 9LT. Telephone: 0044 161 291 2136 Fax: 0044 161 291 2927 Email: [email protected]
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Highlights
A review of the clinical applications of LC-MS/MS glucocorticoid analysis.
The major endogenous glucocorticoids are discussed.
Serum, urine and salivary cortisol are comprehensively covered.
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Non-standard abbreviations
3-HSD = 3-hydroxysteroid dehydrogenase 5THF = Allo-tetrahydrocortisol 5THF = Tetrahydrocortisol 11HSD1 = 11 beta hydroxysteroid dehydrogenase type I 11HSD2 = 11 beta hydroxysteroid dehydrogenase type II 11-DOC = 11-Deoxycortisol ACTH = Adrenocorticotropin hormone AME = Apparent mineralocorticoid excess APCI = Atmospheric pressure chemical ionisation APPI – Atmospheric pressure photo ionisation CBG = Cortisol binding globulin CRH = Corticotropin releasing hormone CRM = Certified Reference Material EQA = External quality assessment ESI = Electrospray Ionisation GC-MS = Gas chromatography-mass spectrometry GC-MS/MS = Gas chromatography-tandem mass spectrometry HPA = Hypothalamic – pituitary – adrenal HPLC = High performance liquid chromatography LC-MS/MS = Liquid chromatography-tandem mass spectrometry LC = Liquid chromatography ONDST = Overnight dexamethasone suppression test
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LLE = Liquid-liquid extraction m/z = mass to charge ratio NEQAS = National External Quality Assurance Scheme PFP = Pentafluorophenyl PPT = Protein precipitation RMP = Reference measurement procedure SLE = Supported liquid extraction SPE = Solid phase extraction THE = Tetrahydrocortisone UFC = Urine free cortisol UPLC = Ultra high performance liquid chromatography
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Abstract Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical technique that offers exceptional selectivity and sensitivity. Used optimally, LC-MS/MS provides accurate and precise results for a wide range of analytes at concentrations that are difficult to quantitate with other methodologies. Its implementation into routine clinical biochemistry laboratories has revolutionised our ability to analyse small molecules such as glucocorticoids. Whereas immunoassays can suffer from matrix effects and cross-reactivity due to interactions with structural analogues, the selectivity offered by LC-MS/MS has largely overcome these limitations. As many clinical guidelines are now beginning to acknowledge the importance of the methodology used to provide results, the advantages associated with LCMS/MS are gaining wider recognition. With their integral role in both the diagnosis and management of hypo- and hyperadrenal disorders, coupled with their widespread pharmacological use, the accurate measurement of glucocorticoids is fundamental to effective patient care. Here, we provide an up-to-date review of the LC-MS/MS techniques used to successfully measure endogenous glucocorticoids, particular reference is made to serum, urine and salivary cortisol.
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Introduction Glucocorticoids are steroid hormones that exert their physiological effects through an interaction with complementary glucocorticoid receptors. These receptors are expressed within almost all human cells. Their stimulation initiates a signalling cascade that terminates in the activation of genes that help regulate normal development and control day-to-day homeostasis. The synthesis, metabolism and the pleiotropic actions of glucocorticoids have been extensively reviewed [1, 2, 3]. Similarly, the clinical indications for their measurement have been outlined in international guidelines [4, 5] and recommendations [6].
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique that has transcended research and analytical chemistry laboratories into routine clinical diagnostics [7]. The exquisite sensitivity and specificity LCMS/MS offers is however reliant on the optimisation of several processes.
This review aims to provide the reader with an up-to-date account of LCMS/MS endogenous glucocorticoid measurement. The optimisation of all relevant LC-MS/MS processes including: sample preparation, mobile phase composition, chromatographic separation and MS/MS detection are considered in this review, with particular reference made to serum, urine and salivary cortisol quantification.
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Endogenous glucocorticoids In humans, the major circulating glucocorticoid is cortisol. This can be measured in many biological specimens including: whole blood, plasma, serum, urine, saliva and hair. This variety of sample types serves many different purposes from clinical diagnosis, research studies and athletic drug screening, with each matrix offering its own analytical advantages and challenges. However, in routine clinical biochemistry, only serum, urine and saliva have found prominence with their use is endorsed for the assessment of the hypothalamic-pituitary-adrenal (HPA) axis [4,5,6].
Serum/plasma cortisol Cortisol is one of the best characterised analytes routinely measured in clinical laboratories. Since 1973, the National Institute for Standards and Technology have made available cortisol standard reference material to promote assay standardisation. Subsequently, several reference measurement procedures (RMPs) have been described that utilise this reference material for calibration. These methods have been used to produce higher-order certified reference materials (CRMs) that are metrologically traceable to SI units [8].
The majority of these RMPs have been validated using isotope dilution (ID) gas chromatography-mass spectrometry (GC-MS). However, advances in sample preparation, liquid chromatography and mass spectrometry have
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heralded a second generation of LC-MS/MS reference systems. The first of these was described in 2004 and utilises sequential off-line solid phase extraction (SPE) and liquid-liquid extraction (LLE) for sample preparation prior to LC-MS/MS analysis [9]. More recently, an on-line SPE method has been described [10] and our own group have developed a candidate RMP using supported liquid extraction (SLE) technology for sample clean-up [11].
Although RMPs offer accurate and precise results, they do not represent a timely and cost-effective option for routine analysis. Instead, they are used to assign materials with a target value so they can serve as certified reference materials (CRMs) to calibrate commercial immunoassay kits [12]. However, despite the availability of serum cortisol RMPs and CRMs, there remains a problem with the standardisation of routine commercial immunoassays. External quality assessment (EQA) schemes provide laboratories with a means to review the analytical performance of their method relative to that of other users. Whereas the intra-method variation for a given sample may be relatively small, it can be significantly increased when all methods are accounted for. The report below provides an example of this variation (Figure 1).
[Insert Figure 1 here.]
This longstanding poor performance has been attributed to matrix effects and poor specificity [13, 14]. Consequently, these assays present a potential risk to accurate patient diagnosis and management. This has provided a
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requirement for robust and accurate cortisol methods in routine clinical laboratories. These requirements have been fulfilled by both LC-MS/MS and gas chromatography coupled to either a single quadrupole mass spectrometer (GC-MS) or a triple quadrupole mass spectrometer (GC-MS/MS). However, whereas serum/plasma cortisol analysis by GC-MS and GC-MS/MS has predominantly been limited to RMPs, LC-MS/MS has been favoured for routine clinical analysis. This may be explained by the general requirement to derivatise non-volatile compounds such as cortisol to make them amenable to GC analysis [8]. Despite improving sensitivity, derivatisation can extend sample preparation time and increase total analytical error. Moreover, it has been suggested that the conditions used for derivatisation can hydrolyse conjugates and metabolites to produce the analyte of interest, thereby reducing analytical specificity [15]. Nevertheless, GC-MS and GC-MS/MS still hold an advantage over LC-MS/MS owing to their higher chromatographic resolution, they therefore remain powerful techniques for specialised applications such as urine steroid profiling [16]. Conversely, LC-MS/MS assays do not generally require derivatisation, are typically higher-throughput and now have comparable sensitivity and specificity to GC-MS or GC-MS/MS. Consequently, their application has generally been favoured for routine clinical diagnostics.
Several LC-MS/MS methods have been used to successfully quantify serum cortisol (Table 1). Of those listed, sample volume requirements range from 25 to 1000 µL, with larger volumes generally necessary to improve assay
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sensitivity. Accordingly, many of these assays are designed to quantify a panel of endogenous and exogenous glucocorticoids, many of which are present in significantly lower concentrations than that of cortisol. However, in routine clinical practice, it is notable that serum and plasma are not always of sufficient volume for the multitude of tests that are typically required. This can be especially true of paediatric samples. Hence, for serum/plasma cortisol analysis, small sample volumes are generally preferable.
Sample pre-treatment and extraction is variable across methods (Table 1). The choice of sample preparation may be influenced by a number of factors including: the relative abundance and physiochemical properties of the analyte of interest, sample matrix, potential interferences, the presence of ion suppression or enhancement, the sensitivity required, the time in which the result needs to be issued, operator proficiency and financial considerations. For serum cortisol, successful sample clean-up has been achieved in a number of ways including: protein precipitation (PPT) [17, 18, 19], LLE [20, 21, 22, 23, 24], SPE [25, 26, 27] and also using a combination of these procedures [28, 29, 30].
Collectively, these approaches aim to remove protein and other potential interferences. Deproteinisation reduces the risk of blocking frits, injectors, columns or narrow bore HPLC tubing and helps reduce matrix effects which can have detrimental effects on analysis [31, 32]. In addition to protein, salts and phospholipids can co-elute with the analyte of interest thereby altering the ionisation efficiency. This can lead to in-source competition for ionisation
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resulting in ion suppression or enhancement [31]. This in turn may compromise the accuracy of the final result. Sample deproteinisation is particularly relevant to serum/plasma cortisol analysis. At present, all current clinical guidelines advocate total cortisol measurement [4,5]. Since cortisol circulates as both free (5%) and protein bound (95%) fractions, it is only through liberating cortisol from its binding proteins that allows its total concentration to be reliably quantified.
PPT methods rely on the addition of a precipitant to liberate cortisol from its binding proteins. Several options are available with metal ions [17] and organic solvents [18,19] popular choices. The relative merits and limitations of different forms of PPT have been extensively tested [33]. PPT methods are rapid, amenable to automation, and relatively inexpensive. However, as a consequence of the required sample dilution, assay sensitivity is reduced. Nevertheless, for cortisol alone, this does not appear to jeopardise the utility of the assay since LOQs below current clinical requirements are still achievable, even with sample volumes as small as 25 µL [17].
Conversely, as both LLE and SPE remove more matrix effects and concentrate the sample, both sensitivity and specificity are improved. These extraction techniques have therefore been favoured for multiplexed assays where additional sensitivity and specificity may be required [20, 21, 22, 23, 25, 26, 27, 28, 29, 30]. However, relative to protein precipitation, LLE and in particular SPE, are technically more demanding, require a longer preparation
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time and are more expensive. For these reasons they are not always practical for routine clinical work.
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Table 1
Analyte(s)
Sample volume (L)
Preparation
Column
Mobile phase (organic/buffer/modifier)
Run time (mins)
Ionisation
LOQ (nmol/L)
Reference
Cortisol
25
PPT (Zinc sulphate / Methanol)
C8
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid
2.6
ESI +
12.5
[17]
Cortisol
250
LLE (Ethyle acetate)
C18
Methanol / 1 mmol/L ammonium chloride / NA
8
ESI -
1.38
[24]
Cortisol, Cortisone
100
LLE (MTBE)
Phenyl
Methanol / NA / NA
3
APPI +
13.76 (Cortisol) 13.87 (Cortisone)
[20]
Cortisol, Cortisone
100
LLE (Ethyle acetate)
C18
Methanol / 5 mmol/L ammonium acetate / NA
Not Stated
ESI +
2.75 (Cortisol), 6.94 (Cortisone)
[23]
Cortisol, Dexamethasone
1000
PPT (Acetonitrile) / SPE (C18)
C18
Methanol / NA / 0.2% formic acid
Not Stated
ESI +
< 20.0 (Cortisol)
[29]
Cortisol, Cortisone, Dexamethasone
200
SPE (Strata X)
Aromax
Acetonitrile / NA / 0.05% formic acid
Not stated
APCI +
0.8 (Cortisol) 0.8 (Cortisone)
[25]
Cortisol, Dexamethasone, Prednisone, Prednisolone
500
SPE (HLB)
C18
Methanol / 5 mmol/L ammonium acetate / NA
6
ESI -
14.85 (Cortisol)
[26]
Cortisol, Cortisone, Prednisolone, Prednisone
50
PPT (Acetonitrile), evaporation, reconstitution
Phenyl
Acetonitrile / NA / 0.1% formic acid
8
ESI +
2.76 (Cortisol), 1.4 (Cortisone)
[18]
Cortisol, Cortisone, 17-OHP, 21-DOC, 11-DOC, Deoxycorticosterone
500
LLE (Methylene chloride)
C18
Methanol / NA / NA
10
ESI +
2.75 (Cortisol),* 13.87 (Cortisone)* 14.4 (11-DOC) *
[21]
Cortisol, Cortisone, Prednisolone, Dexamethasone, 11-DOC,
250
SPE (HLB)
C18
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid
3
ESI +
3.75 (Cortisol), 3.75 (Cortisone), 5.00 (11-DOC)
[27]
Cortisol, Cortisone, Dexamethasone, Prednisolone, Prednisone, Methylprednisolone
500
PPT (Acetonitrile) / LLE (Dichloromethane)
C18
Methanol / 2mmol/L ammonium acetate / 0.1% formic acid
12
ESI +
8.3 (Cortisol), 8.3 (Cortisone)
[28]
Cortisol, Cortisone, Testosterone, Dihydrotestosterone, Progesterone
100
PPT (Methanol)
C18
Methanol / NA / 0.1% formic acid
12
ESI +
5.24 (Cortisol), 0.83 (Cortisone)
[19]
85
LLE (Ethyl acetate:hexane, 80:20 (v/v))
C18
Acetonitrile / NA / 0.1% formic acid
6.1
ESI -
1.95 (Cortisol), 1.58 (Cortisone), 0.098 (11-DOC)
[22]
500
PPT (HCl) / SPE (HLB)
C18
Methanol / 5 mmol/L ammonium acetate / NA
Not stated
ESI -
9.93 (Cortisol)
[30]
Cortisol, Cortisone, 11-DOC, Dexamethasone, Prednisolone, Prednisone, Testosterone, Androstendione, Progesterone Cortisol, Dexamethasone, Methylprednisolone, Prednisone, Prednisolone, Mycophenolic acid, Mycophenolic acid glucuronide
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There is marked heterogeneity in mobile phase composition across the methods listed in Table 1. Whereas water is typically used for the aqueous phase, the choice of organic solvent varies between methanol and acetonitrile. As a protic solvent, methanol can promote [M+H]+ ion formation. However, when mixed with water, its viscosity can increase resulting in a high column backpressure. This is generally undesirable and can result in column deterioration or system leaks. Since the advent of UPLC, there has been a trend towards using sub 2 micron particle columns as they can enhance peak resolution. Our experience has found these columns to be especially useful for steroid analysis as they can help separate isobaric interferences [34]. However, columns with < 2 m particles can be susceptible to rapid degradation when subjected to very high backpressures. This can result in deterioration of chromatographic separation and often necessitates that low flow rates are used which consequently increases injection-to-injection time. Acetonitrile offers a useful alternative to methanol as it has a lower viscosity when mixed with water and therefore produces lower backpressures. However, as an aprotic solvent, acetonitrile does not enhance [M+H]+ ion formation, is generally more expensive and, at times, of limited availability.
While several methods do not make use of a buffer [18, 19, 20, 21, 22, 25, 29], others have used ammonium acetate [17, 28, 26, 27, 30, 23] or ammonium chloride [24]. The presence of a buffer can improve the ionisation of cortisol, this may be through adduct formation (e.g. ammonium chloride) or through enhancing in-source ionisation (e.g. ammonium acetate). As a neutral molecule, control of pH has minimal effect on cortisol analysis however,
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formic acid has been used to enhance ionisation both in the positive and negative ionisation modes [17, 18, 19, 22, 25, 28, 29, 27]. In the positive ionisation mode, formic acid can provide a rich supply of protons which favour the formation of [M+H]+ ions. Conversely, in the negative ionisation mode, cortisol can form a specific adduct with formic acid as discussed below. In our experience, it is good practice to experiment with different mobile phase compositions to optimise ionisation. In all cases, we have found that LC-MS grade solvents, buffers and modifiers produce the best results. These are typically purer than their HPLC- or reagent-grade equivalents.
By virtue of its hydrophobicity (Log P = 1.43 [35]) and steric properties, cortisol can be chromatographically separated from structurally analogous compounds by traditional alkyl bonded reverse phases. Several C18 columns are now available with differences in end-capping, carbon load and ligand surface area coverage just some of the factors that contribute to the varying performance between columns and manufacturers. Other than C18 columns, a C8 phase has successfully been used [17]. C8 columns rely on similar hydrophobic interactions however, owing to their shorter carbon chain length, analytes are less avidly retained thus permitting shorter run-times. The use of phenyl columns have also been described [18, 20, 25]. This mode of separation relies on pi-pi interactions between aromatic compounds and the stationary phase. Phenyl ligands are therefore capable of exploiting the aromatic ring system present in cortisol to chromatographically separate it from structurally similar compounds. This has reportedly helped separate cortisol from the prednisolone metabolite tetrahydroprednisolone, a potential
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isomeric interference [20]. However, in two of the assays developed, it is unlikely that full advantage of the phenyl phase is taking place [18, 25]. In these methods, acetonitrile has been used in the mobile phase. Acetonitrile is known to delocalise the electron clouds that help form pi-pi interactions, therefore suggesting the separations observed involve other retention mechanisms.
Although all assays listed achieve an LOQ below any current clinical requirements, the method described by Ray et al is notably lower than the others (0.80 nmol/L) [25]. This method uses SPE for sample purification and atmospheric pressure chemical ionisation (APCI) to convert cortisol into an ion prior to MS/MS detection. APCI relies on the vaporisation of the column eluent by a heated nebuliser, the remaining molecules (including the analyte of interest) are subsequently ionised by a corona discharge needle. This produces stable ions which then enter the mass spectrometer. APCI is particularly efficient at enhancing the ionisation of compounds that ionise less effectively in solution such as cortisol [36]. This efficiency has contributed to APCI generally being considered a harder form of ionisation than alternatives. Indeed, it has been suggested that APCI can cause an exchange between the deuterated atoms of labelled internal standards with the hydrogen atoms of water therefore compromising accurate quantification [37]. This observation has also been refuted, suggesting instrument-specific differences may effect ionisation [36]. In a novel approach, one cortisol method has used atmospheric pressure photoionisation (APPI) [20]. APPI uses ultraviolet light to irradiate the column eluent which initiates a cascade of reactions resulting
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in the formation of stable ions which are channelled into the mass spectrometer. Other than APCI and APPI, the majority of methods have used ESI to ionise cortisol. In ESI, the column eluent enters a stainless steel capillary to which a voltage is applied. A heated gas, usually nitrogen, is passed along the capillary resulting in the nebulisation of the eluent and production of charged droplets. These droplets undergo coulombic fission and are converted into stable ions through desorption and desolvation before entering the mass spectrometer. Relative to APCI and APPI, ESI in the positive ionisation mode does not remove as many matrix effects [37, 38]. However, if careful attention is paid to sample preparation and chromatography, this is not problematic for serum/plasma cortisol analysis since all ESI methods listed in Table 1 achieve sufficient performance characteristics to be clinically useful.
Despite the variation in preparation, separation and ionisation, the transitions used to detect cortisol are similar across methods. To complement this section of the review please refer to Figure 2. This shows a precursor ion scan in the positive ionisation mode of a 1 mg/L methanolic solution of cortisol infused into a Waters Acquity TQD™ LC-MS/MS system under the conditions described by Owen et al [17] (Fig. 2A). Here, the m/z 363 peak corresponding to the [M+H]+ cortisol ion is clearly visible, it is this ion that is scanned for in the first quadrupole of all methods using the positive ionisation mode. In figure 2B we present the product ion scan acquired from applying a collision energy of 28 eV to the precursor ion in the collision cell. In addition,
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we have included the corresponding theoretical fragments for the common product ions (Fig. 2B).
The majority of assays listed in Table 1 have favoured the quantification of cortisol using the m/z 363>121 transition [20, 23, 25, 27, 28, 29]. This corresponds to detection of the A-ring fragment following cleavage of the adjoining B-ring bonds [39]. When optimised, the m/z 363>121 is generally the most sensitive transition however, it is not always specific with interference reported from prednisolone and its metabolites [11, 17, 18] (Figure 3). Conversely, the m/z 363>327 [18, 19] and m/z 363>97 [17] transitions have been found to be more selective yet not as sensitive. Whereas the m/z 363>327 transition corresponds to cleavage of two hydroxyl groups, the m/z 363>97 transition represents cleavage and subsequent rearrangement on the A-ring fragment [40, 41]. In addition, Ray et al have also used m/z 363>267 as a secondary transition.
[Insert Figure 2 here]
[Insert figure 3 here]
Whereas ESI in the positive ionisation mode is favoured for cortisol analysis, four of the methods have used ESI in the negative ionisation mode [22, 24, 26, 30]. Of these, two have exploited of the ability of cortisol to form an adduct
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in this mode. Huang et al have used ammonium chloride to form a [M+35]- precursor ion and subsequently scanned for the m/z 397>35 product ion following fragmentation of cortisol and the chloride ion in the collision cell [24]. This allows for a very sensitive method however; it is notable that scanning for product ions in the lower range of the mass scale is generally not recommended as specificity can be compromised. The method described by Methlie et al has instead monitored the formic acid adduct [M+46] - and the corresponding m/z 407>297 transition following collision induced dissociation [22]. Conversely, two methods do not rely on adduct formation and instead scan for the native [M-H]- ion which fragments to [M-H-CH2O]-, this corresponds to m/z 361>331 [26, 30]. Relative to ESI in the positive ionisation mode, detection of cortisol in the negative ionisation mode has been reported to improve signal-to-noise [26, 30].
Interestingly, four of the methods summarised have used structural analogues as an internal standard including: flumethasone [26, 30], prednisolone-d6 [28] and 6-methylprednsiolone [24]. To ensure the internal standard provides true compensation for variability in ionisation, its retention time should be approximately equal to that of the analyte of interest. Therefore, given their widespread availability, it is highly recommended that isotopically labelled internal standards are used in preference to structural analogues for assay validation.
In general, short run times are desirable for a prompt result turnaround. The assay developed by Owen et al has been specifically designed for
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implementation into a routine clinical laboratory [17]. The other assays listed in Table 1 are mainly multiplexed and designed to ensure the adequate separation of all compounds scanned for. As such, they require longer run times.
One application where LC-MS/MS serum cortisol quantification has been found to be increasingly important is in patients prescribed metyrapone. This 11-hydroxlase inhibitor is used to block cortisol synthesis in patients with Cushing’s syndrome. In addition, it is used in some countries to help diagnose adrenal insufficiency. Serum cortisol measurement by routine immunoassays has been found to be prone to interferences in samples collected from patients prescribed metyrapone [42, 43]. Inhibition of 11- -hydroxlase can cause gross elevations in cortisol precursors (e.g. 11–deoxycortisol) that can interfere with immunoassay cortisol quantification therefore potentially masking hypoadrenalism [43, 44]. As metyrapone dosing regimens may be titrated against cortisol concentrations this has important ramifications on patient care. This fact has been recognised by the Endocrine Society in their recent clinical practice guideline concerning Cushing’s Syndrome treatment [44]. In this document, LC-MS/MS is recommended for serum and urine cortisol quantification in patients prescribed metyrapone as it circumvents the problems associated with immunoassays. McWhinney et al also suggest that when metyrapone is administered as part of a dynamic function test for the diagnosis of adrenal insufficiency, concomitant measurement of 11deoxycortisol along with cortisol can aid result interpretation [27].
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Urine free cortisol and its metabolites Urine free cortisol (UFC) is advocated for the investigation of hypercortisolism because it represents the non-protein bound cortisol fraction that is freely filtered at the glomerulus [4]. Whereas immunoassays suffer from interference due to cross reactivity with cortisol A-ring metabolites and other steroids, the selectivity and sensitivity of LC-MS/MS overcomes this to provide accurate results [45]. Consequently, many urinary free cortisol LC-MS/MS methods have been published (Table 2).
Urine cortisol measurements are only recommended following a 24h collection period [4], hence obtaining sufficient sample volume is rarely problematic. This is reflected by the relatively larger volumes that are used in urine assays compared to that of serum (Table 2).
Two of the methods listed make use of the simplest form of sample preparation namely ‘dilute-and-shoot’ [46, 47]. This can significantly reduce both preparation time and inter-analyst variation. However, this is at the expense of introducing relatively dirty samples into the LC-MS/MS system. This can be detrimental as the risk of blocking tubing or frits increases, column lifetime can reduce, and the mass spectrometer itself may require additional services as salts can deposit within the system. Although more comprehensive sample clean-up is recommended, analysts wishing to explore dilute-and-shoot should at least centrifuge the specimen prior to sampling to ensure particulates do not enter the LC-MS/MS system. In an evolution of dilute-and-shoot, Kushnir et al al have used a 2D chromatography system
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with an online C18 guard cartridge to trap cortisol [36]. In this method, the sample is first injected onto a guard column where it is trapped and subjected to an organic wash. This wash is sufficiently organic to remove hydrophilic interferences to waste without disrupting the interaction between the analyte of interest and the guard column phase. After the sample is suitably purified, the flow path is switched and the guard column eluent is diverted to the analytical column. The organic composition of the mobile phase is then increased to liberate the analyte of interest from the guard column onto the analytical column. This allows further sample clean-up in the form of traditional reverse-phase chromatography prior to MS/MS detection.
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Table 2 Analyte(s)
Sample volume (L)
Preparation
Column
Mobile phase (organic/buffer/modifier)
Run time (mins)
Ionisation
LOQ (nmol/L)
Reference
Cortisol
500
dilution (1:1 water)
C18
Methanol / NA / 0.003% trifluoroacetic acid
3
APCI +
13.8
[46]
Cortisol
500
Online C18 cartridge
C18
Methanol / 5 mmol/L ammonium formate / NA
8
APCI +
5.5
[36]
5
ESI +
21.5
[48]
4.5
ESI +
2
[38]
8
ESI -
1.38 6. 0 (Cortisol), 6.0 (Cortisone)
[24]
Cortisol
100
PPT (trichloroacetic acid)
C18
Cortisol
1000
SPE (HLB)
C18
Cortisol
250
LLE (ethyl acetate)
C18
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid Methanol / 2mmol/L ammonium acetate / 0.1% formic acid Methanol / 1 mmol/L ammonium chloride / NA
Cortisol, Cortisone
500
LLE (methylene chloride)
C18
Methanol / NA / NA
3
ESI +
Cortisol, Cortisone, 6-sulfatoxymelatonin
250 (minimum)
dilution (1:1 water)
C18
Acetonitrile / NA / 0.1 % formic acid
6
ESI +
3.8 (Cortisol) * 4.7 (Cortisone) *
[47]
Cortisol, Cortisone
100
Turboflow
C18
Methanol / NA / NA
9.5
APCI +
5.5 (Cortisol) 2.8 (Cortisone)
[54]
Cortisol
300
SPE (C8 Column)
C18 monolithic
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid
4.5
ESI +
5
[50]
Cortisol, Cortisone
1000
SPE (MAX)
C8
Methanol / 20 mmol/L ammonium formate / NA
10
ESI +
0.4 *
[51]
Cortisol, Cortisone, Tetrahydrocortisol, Allotetrahydrocortisol, Tetrahydrocrotisone Cortisol, Cortisone, Tetrahydrocortisol, Allotetrahydrocortisol, Tetrahydrocrotisone Cortisol, Cortisone, Tetrahydrocortisol, Allotetrahydrocortisol, Tetrahydrocrotisone
1000
SPE (HLB)
C18
Methanol / NA / 0.1% formic acid
18
ESI +
2000
SPE (HLB) / dilution (1:1 water)
Biphenyl
Methanol / NA / 0.1% formic acid
19.5
ESI -
480
PPT (acetonitrile)
C18
25
ESI -
Acetonitrile / 5 mmol/L ammonium acetate / 0.1% formic acid
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0.3 (Cortisol) 0.3 (Cortisone) 2.7 (Tetrahydrocortisol) 2.7 (Allo-tetrahydrocortisol) 2.3 (Tetrahydrocortisone) 8.3 (Cortisol) 1.7 (Cortisone) 0.8 (Tetrahydrocortisol) 0.8 (Allo-tetrahydrocortisol) 1.6 (Tetrahydrocortisone) 0.14 (Cortisol) 0.14 (Cortisone) (Tetrahydrocortisol) (Allo-tetrahydrocortisol) (Tetrahydrocortisone)
[37]
[52]
[53]
[49]
Alternative approaches to sample preparation have involved protein precipitation with trichloroacetic acid [48] or acetonitrile [49], LLE using ethyl acetate [24] or methylene chloride [37] and SPE [45, 50, 51, 52, 53]. With its enhanced purification providing improved sensitivity and specificity, SPE is the favoured approach. The advantages SPE confers are highlighted by the lower limits of quantification achieved using this technique relative to others. Although few authors disclose any preliminary work that influenced their choice of SPE phase, Fong et al evaluated their method using both a mixed mode anion exchange (MAX) phase and a mixed mode cation exchange (MCX) phase, superior recovery and imprecision were observed using the MAX cartridges [51]. In a novel approach, Sánchez-Guijo et al have introduced samples directly into a Turboflow™ chromatography system [54]. This uses turbulent solvent flow in a short chromatography column packed with porous particles to separate smaller molecules from larger ones (e.g. proteins) in complex biological matrices. Whereas the small molecules interact with particle pores, larger ones are size-excluded and discarded. Once separated, an analytical column can be used for improving separation of the smaller molecules prior to MS/MS analysis. The use of the Turboflow™ dramatically reduces bench-time preparation and inter-operator variability.
The majority of methods listed in Table 2 achieve chromatographic separation of cortisol using the hydrophobic interaction of C8 and C18 columns. However, in two original approaches, one method makes use of a monolithic phase [50] whereas another utilises a biphenyl column [53]. Monolithic phases rely on a silica based porous rod system to achieve chromatographic
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separation. The stability of these porous rods allow higher flow rates (up to 1 mL/min) without the consequential rise in pressure that is observed with particle based chromatography. This permits the development of efficient and rapid methods. Conversely, the biphenyl phase, like its phenyl counterpart, relies on the pi-pi interaction between hydrophobic aromatic compounds and the biphenyl ligand, this can produce exceptional separation of structurally similar compounds such as the stereoisomers tetra-hydrocortisol and allotetra-hydrocortisol [53].
Although most assays have used an isotopically labelled internal standard, one method has used 6--methylprednisolone to compensate for variation in ionisation [47]. Interestingly, another method evaluated methylprednisolone as an alternative for cortisol-d4 [53]. The results demonstrated that the structural analogue was 10-15% less accurate than the isotopically labelled internal standard for the quantification of cortisol and its metabolites.
Whereas ESI in the positive ionisation mode is the most common approach, three methods have used ESI in the negative ionisation mode [24, 49, 53]. Under these conditions, Cuzzola et al specify that the formic acid adduct [M+HCOO]- consistently fragments to [M-H-CH2O]- for all analytes providing high sensitivity and specificity [53]. In contrast, three methods have utilised APCI in the positive ionisation mode, this reportedly increased signal-to-noise by 2-3 fold compared to ESI [36, 46, 54]. Despite the differences used to achieve ionisation, all methods have determined an LOQ within the desirable clinical range for routine UFC measurements. The differences observed for
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ionisation may be directly related to the buffers and modifiers used, or the insource ionisation which can vary between manufacturers
Aside from hypercortisolism, quantification of urine cortisol and its metabolites can also be effective in the diagnosis of other pathologies related to disordered steroid production or metabolism. Several enzymes are involved in modulating the availability, metabolism and clearance of cortisol (Figure 4) [55]. Hepatic and adipose tissue both express the enzyme 11-hydroxysteroid dehydrogenase type I (11HSD1) which converts the inactive cortisone to the active cortisol. This reaction can also be reversed in the presence of 11hydroxysteroid dehydrogenase type II (11HSD2) which is expressed in renal and colon cells. Furthermore, the action of the hepatic A-ring reductases (5 and 5reductase), in conjunction with 3-hydroxysteroid dehydrogenase (3-HSD), inactivate cortisol to the tetrahydrometabolites: allotetrahydrocortisol (5THF), tetrahydrocortisol (5THF), and tetrahydrocortisone (THE). Thus, as conjugation is predominantly a hepatic process, the ratio of cortisol and cortisone tetrahydrometabolites (i.e. 5THF + 5THF / THE) provides an index of hepatic 11HSD1 activity. Similarly, the free cortisol:cortisone ratio provides information on the activity of 11HSD2. In addition, the ratio of 5THF / 5THF reflects the relative activities of global 5 and 5reductase activity [56, 57].
[Insert Figure 4 here]
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Much of the early work measuring these metabolites was undertaken using GC-MS with the syndrome of apparent mineralocorticoid excess (AME) one of the first conditions identified [58]. AME is characterised by a congenital defect in 11HSD2 which prevents the oxidation of cortisol to cortisone. Without this protective mechanism, cortisol can accumulate and stimulate the mineralocorticoid receptor (MR). Interestingly, AME can also be acquired through inhibition of 11HSD2 secondary to carbenoxolone therapy or excessive consumption of glycyrrhizinic acid (commonly found in liquorice). Unregulated MR stimulation results in sodium and water retention consequently leading to hypertension. Two congenital forms of AME have been described, both of which can be determined by virtue of an elevated urine free cortisol to cortisone ratio [59]. However, Type II is distinguishable from Type I as A-ring reduction is the major deficiency [60, 61].
In addition to AME, GC-MS has helped to identify glucocorticoid metabolic imblances in patients with glucocorticoid suppressible aldosteronism and adrenal adenomas [62, 63], females with polycystic ovarian syndrome [64], obesity [65], and congenital adrenal hyperplasia secondary to 21-hydroxylase deficiency [66]. Furthermore, a debate has recently emerged in the literature suggesting that the cross-reactivity observed with cortisol metabolites in immunoassays may actually increase their diagnostic sensitivity for Cushing’s Syndrome [67]. Therefore, to further increase the diagnostic sensitivity of LCMS/MS it may be necessary to measure cortisol and its metabolites to provide a complete assessment of hypercortisolism. Collectively, this evidence has
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provided an impetus for the development of LC-MS/MS assays capable of measuring cortisol and its metabolites.
Consequently, several of the assays listed in Table 2 have been developed to measure cortisone concomitantly with cortisol [37, 47, 49, 51, 52, 53, 54]. In comparison to cortisol, cortisone differs by the presence of a C=O group in position 11. Taylor et al suggest that the presence of this bond may be responsible for the different ionisation pattern observed with cleavage of the C-ring (as opposed to the B-ring with cortisol) producing the most abundant fragment m/z 361 > 163 when analysed in the positive ionisation mode [37]. Interestingly, as prednisolone and cortisone share the same molecular weight (360.44 Da) crosstalk has also been reported in their analysis [22, 28]. This can be prevented by ensuring adequate chromatographic separation of the two compounds.
In normal adults, approximately twice as much cortisone is excreted compared to cortisol, as such sensitivity has not proved problematic. Hence, it has been suggested that the the cortisol:cortisone ratio can add value to result interpretation and the evaluation of 11HSD2 activity [68].
More recently, several LC-MS/MS assays have been designed to provide a more complete picture of cortisol metabolism through measurement of cortisol, cortisone and their tetrahydrometabolites: 5THF, 5THF and THE. One of the earliest methods published quantified only the tetrahydrometabolites and required a derivatisation procedure that increased
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sensitivity 15-80-fold [69]. Campino et al have since published a method using an F5 column that did not allow for chromatographic separation of the stereoisomers 5THF and 5THF and therefore provided limited information regarding the 5 and 5reductase activity [70]. F5 columns act as a lewis acid due to the presence of 5 electronegative fluorine groups. This ensures the phenyl ring is electron deficient which can help separate closely related ring systems such as those present in steroids. However, care is still required to ensure the gradient encourages separation of closely related compounds such as stereoisomers. More recently a series of methods have been published that achieve separation of all compounds including the 5THF and 5THF [49, 52, 53, 71]. Interestingly, two of the methods have favoured ESI in the negative ionisation mode owing to its enhanced signal-to-noise relative to ESI in the positive ionisation mode [53] However, this observation may be instrument-specific and it is always worth experimenting in both modes. Importantly, the cycle times injection-to-injection for these three assays range from 18 – 25 minutes [49, 52, 53, 71], run times this long are not always conducive with routine, high-throughput laboratories. Another potential problem with these assays is they are unable to truly compensate for variation in ionisation due to a lack of availabile isotopically labelled internal standards. Taking the method of Zhai et al as an example, here the structural analogue methylprednisolone elutes after approximately 6 minutes, the analytes of interest subsequently elute between 8 and 18 minutes [49]. Thus, it is apparent that there is a significant period where no allowance is made for variation in ionisation efficiency. In an original approach, Marcos et al have
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used a C18 column to achieve excellent separation of a panel of 67 corticosteroids including many additional glucocorticoid metabolites [71].
Salivary cortisol By virtue of its lipophilic nature, serum free cortisol can passively diffuse into saliva via the acinar cells in a process that is independent of salivary flow rate [72]. Therefore, like UFC, salivary cortisol quantification provides an index of serum free cortisol concentration [73]. The use of saliva for cortisol measurement has several advantages over both urine and serum: as a matrix itself, saliva contains relatively fewer interfering compounds and in that regard presents less of an analytical challenge. In addition, its non-invasive, costeffective collection is appealing to both patients and clinicians. Consequently, advances in sampling techniques coupled with a strong evidence base have propelled salivary cortisol from a novel assay confined to endocrine and psychological research into routine clinical practice. Indeed, it is now recommended as a first-line diagnostic test in the investigation of Cushing’s Syndrome [4]. Its transition into clinical laboratories has in part been driven by LC-MS/MS with accurate quantification of low salivary cortisol concentrations now possible. Originally measured by RIA and immunoassay, salivary cortisol has been reported to suffer from non-specific cross-reactivity with endogenous and exogenous glucocorticoids. In addition, poor standardisation between assays has contributed to assay-specific variation meaning results are not directly comparable [74, 75]. This problem is compounded by the expression of 11HSD2 in the parotid glands which ensures the cortisone:cortisol ratio is significantly higher than in serum.
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The structural similarity of cortisone to cortisol has proved difficult for immunoassays to differentiate between the two. These limitations have largely been circumvented by the application of LC-MS/MS. Table 3 provides a summary of recently published LC-MS/MS salivary cortisol assays.
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Table 3
Analyte(s)
Sample Volume (L)
Preparation
Column
Mobile Phase (organic/buffer/modifier)
Run time (mins)
Ionisation
LOQ (nmol/L)
Reference
Cortisol
100
LLE (dichloromethane)
C18
Methanol / NA / NA
10
ESI -
0.07
[78]
Cortisol
100 / 200
PPT (heat denaturation) /online SPME (Supel Q PLOT capillary)
C8
Methanol / NA / 1% acetic acid
5
ESI +
0.01*
[80]
Cortisol
250
PPT (acetonitrile / acetic acid)
C8
Methanol / NA / 0.5% acetic acid,
5
ESI +
1.4
[79]
Cortisol, cortisone
50
online SPE (C18)
C18 monolithic
Methanol, 0.1% formic acid
4
ESI +
0.75 (Cortisol) 0.75 (Cortisone)
[83]
Cortisol, cortisone
500
PPT / SPE (strata X) / derivitisation
C18 monolithic
Methanol, 500 mmol/L ammonium formate / NA
Not stated
ESI +
0.66 (Cortisol) 2.77 (Cortisone)
[76]
Cortisol, Cortisone
100
online SPE (C8)
C18
Methanol / 2 mmol/L ammonium acetate /0.1% formic acid
5
ESI +
0.39 (Cortisol) 0.78 (Cortisone)
[81]
Cortisol
200 / 100
online SPE (C8)
C18
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid
4
ESI +
2
[82]
Cortisol, Cortisone, Melantonin
50
Turboflow
C18
Methanol, 1.5 mmol/L ammonium formate / 0.1% formic Acid
13
ESI +
0.55 (Cortisol), 5.5 (Cortisone)
[84]
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These methods share a common requirement for small sample volumes ranging from 50-250 L however, one method requires 500 µL [76]. Generally, saliva is not a difficult specimen to obtain, with the availability of dedicated collection vessels promoting standardised collection procedures. Nevertheless, despite following these procedures, salivary yields can be poor in paediatric populations and acutely unwell patients. For this reason, small sample volumes remain desirable for routine analysis.
Salivary cortisol quantification is used to assess the nadir of an individuals diurnal rhythm. Consequently, the cortisol concentration in normal subjects at this time can challenge the functional sensitivity of immunoassays. LC-MS/MS analysis is better suited to this task because it is 10-100 fold more sensitive than RIA and confers greater specificity [77]. To achieve this, LC-MS/MS salivary cortisol methods generally benefit from a sample preparation that purifies and concentrates cortisol prior to analysis. This has been accomplished by various techniques including LLE using dichloromethane [78] and protein precipitation with evaporation of the supernatant prior to reconstitution [79]. Heat denaturation has also been combined with an online solid phase micro extraction (SPME) technique that consists of a 2D chromatography system that loads the sample onto a fused silica capillary [80]. In a similar approach the use of off-line [76] or on-line [81, 82, 83] SPE has been used for sample purification. When performed off-line, SPE can take a relatively long time to prepare with the process heavily reliant on operator proficiency. Conversely, online SPE reduces inter-operator variability and ensures maximum recovery of the analyte as loss is minimal compared to the
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offline evaporation of an eluate. Online SPE using cartridges has advantages over the use of a guard column as the washes can be performed separately to the main flow path hence solvents other than those used in the mobile phase can be utilised for sample clean up [83]. Another approach to online sample clean-up has used Turboflow™ technology to purify the sample prior to introduction to the LC-MS/MS, this requires little sample pre-treatment ensuring minimal inter-operator variation [84].
Two assays have been validated using a monolithic C18 phase [76, 83]. As discussed above, the application of a monolithic phase as opposed to a traditional silica particle based column can facilitate the development of rapid and efficient methods such as that described by Jones et al which achieves separation of cortisol and cortisone within 4 minutes (Figure 5) [83].
[Insert Figure 5 here]
Sensitivity can be crucial in salivary cortisol methods, as such the majority of assays listed in Table 3 use ESI in the positive ionisation mode to monitor the m/z 363>121 transition [81, 82, 83, 84]. Conversely, Jonsson et al have reported observing interference in the m/z 363>121 transition and have instead opted to use m/z 363>309 [79]. Vieira et al use hydroxylamine for derivatisation and scan for the corresponding m/z 393>136. Another method has analysed cortisol in the negative ionisation mode using the m/z 361>331 transition [78]. Finally, one method uses just a single quadrupole to scan for the [M+H]+ ion corresponding to m/z 363 [80]. The use of single quadrupole
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mass spectrometers is generally not recommended, specificity is considerably reduced and therefore the chance of detecting an interfering compound is increased.
Salivary cortisone and cortisol can be measured concurrently [76, 81, 83, 84]. It has been suggested that this can facilitate result interpretation [81]. The non-invasive nature of salivary collection lends itself to the assessment of hydrocortisone replacement therapy whereby day-curves can be produced to ensure patients are provided with optimal steroid replacement. Measuring cortisone and cortisol concomitantly can demonstrate contamination from oral hydrocortisone shown by an unexpectedly high cortisol:cortisone ratio [81]. Perogamvros et al have proposed that the salivary free cortisone concentration may more accurately reflect the concentration of free serum cortisol [85].
Although much work has focused on the application of salivary cortisol in the diagnosis of Cushing’s syndrome, there is evidence to suggest it can also be used in the diagnosis of adrenal insufficiency [81, 86, 87]
Future directions Over the past decade there has been an exponential rise in the number of LCMS/MS glucocorticoid publications. This trend does not appear to be declining with advances in sample preparation, chromatography, and mass
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spectrometry providing scientists with the instrumentation required to improve existing techniques. This, coupled with increasing clinical knowledge of glucocorticoid metabolism, is providing a solid platform to continue advancing the field.
For many scientists and clinicians with an interest in glucocorticoid analysis, the development of robust serum/plasma free cortisol assays remains an important goal. Free hormone quantitation is analytically challenging as analysis itself can disturb the equilibrium between the free and protein bound fractions bringing into question the integrity of the final result. Although free serum cortisol assays have been described, they typically require extensive sample preparation such as ultrafiltration [24, 88] or equilibrium dialysis [89]. These techniques are not conducive to high-throughput laboratories and it is possible that advances in sample preparation or LC-MS/MS methodology could advance free hormone analysis into a routine application. This would require extensive clinical studies to support its utility.
The case for LC/MS/MS measurement of urine cortisol, cortisone and their metabolites has been outlined above. Although initial assays have been described, their cycle times are relatively long when compared to most routine LC-MS/MS methods [49, 52, 53]. Future work is required to develop these assays for routine use. In addition, there is a general requirement for manufactures to provide isotopically labelled internal standards for the glucocorticoid metabolites.
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Another potentially exciting development in the field of LC-MS/MS glucocorticoid analysis is the quantification of hair total cortisol. As cortisol has been found to deposit in the hair shaft, it is postulated that hair cortisol analysis may allow clinicians to assess chronic exposure to cortisol to aid in the diagnosis of Cushing’s Syndrome, monitor hydrocortisone replacement therapy and to assess pathologies associated with chronic stress [90]. Despite the growing evidence base [91, 92, 93, 94], the utility of hair cortisol measurements has yet to be recognised in any clinical guidelines. One possible reason for this is the pre-analytical factors involved with hair collection [95]. Further work is required to fully assess the clinical utility of glucocorticoid hair measurements.
It is also important to appreciate that unlike cortisol, no RMPs currently exist for any other endogenous glucocorticoids or their metabolites. For this reason, metrological traceability cannot be guaranteed and assay standardisation may in future be identified as a problem. Thus, there remains a requirement for RMPs to be developed that are sufficiently accurate and precise they can be used to produce CRMs that will promote assay standardisation.
One of the major advantages LC-MS/MS holds over immunoassay is the ability for laboratories to develop novel assays relatively quickly. With the introduction of new equipment and the recognition of new endogenous metabolites opportunities will continue to be presented for novel assay development.
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Historically, one factor that has limited the wider uptake of LC-MS/MS is the relative expense of the equipment compared to other analysers. As the number of manufacturers has increased and technology has advanced, entrylevel instruments have gradually become more affordable. This has helped a greater number of laboratories adopt LC-MS/MS technology. However, their cost and the expertise required to successfully operate them remain an issue, especially in the developing world. To circumvent this, there is a general requirement for manufacturers to develop equipment that can integrate into routine, high-throughput, automated chemistry analysers that will require minimal operator input.
Summary The field of glucocorticoid quantitation is being propelled by LC-MS/MS. With its adoption by routine clinical chemistry laboratories, there has been increasing awareness of the advantages this powerful technology offers over existing methodologies. This recognition has now moved beyond the laboratory with many clinicians and research scientists cognisant of the advantages LC-MS/MS offers for patient diagnosis and management as well as for high impact clinical research.
Despite its growing reputation, there have been general concerns raised regarding LC-MS/MS analysis. Many of these centre on the variability observed across methods that are designed to measure the same analyte [96, 97, 98, 99]. This concern has been justified in this review. Although many methods rely on established validation criteria (e.g. FDA IVD guidelines [100])
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there is widespread variation to the approaches used with many of these, especially the older ones, reliant on inadequate validation.
Several of the methods reviewed have used structural analogues as opposed to a stable, isotopically labelled internal standard. This can be problematic if the internal standard and analyte of interest do not co-elute. To compensate for variation in ionisation efficiency, it is essential that the physiochemical properties of the internal standard resemble those of the analyte of interest as closely as possible. Although isotopically labelled internal standards are recommended over structural analogues, problems can still be encountered. Duxbury et al have shown that when using cortisol-d2 as an internal standard to measure cortisol, the naturally occurring isotope cortisol-365 can falsely increase the internal standard concentration therefore reducing the relative response for cortisol [101]. This can largely be avoided by selecting internal standards with more than 2 substituted deuteriums. Davison et al have also reported that the location of the deuterium labels can be crucial as some sites will back-exchange with hydrogen atoms thereby causing a reduction in internal standard area and an increase the relative response of the analyte [102]. Many of the concerns surrounding internal standards have been addressed with the introduction of superior C13 based compounds [38].
When clinical samples are to be measured, the assay should be validated using samples taken using blood collection tubes from several manufacturers and using different anticoagulants. These could all effect how well the analyte of interest ionises because of differing ionisation efficiency profiles. Matrix
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effects can be evaluated using infusion experiments to qualitatively test for ion suppression [31] and also quantitatively by comparing the recovery of spiked analyte from aqueous and plasma samples [103]. It is also possible to monitor interferences directly using targeted analysis of individual phospholipids [104].
Another common problem is the investigation of analytical interference. Method validation typically includes analysis of a panel of endogenous and exogenous compounds structurally analogous to the analyte of interest. The potential interferent is normally added in excess to a solution and injected into the LC-MS/MS system, any interference is deemed significant if a signal greater than the LOQ of the assay is detected in any of the m/z transitions scanned for. Although sufficient for endogenous compounds, this approach is not comprehensive enough for exogenous compounds since it is often parent compound metabolites that manifest as an interferent. For example, although prednisolone itself can be separated from cortisol, its metabolites have been reported to interfere in LC-MS/MS analysis [11,17, 18]. As it is not always possible to investigate drug metabolites, it is best practice to develop a method capable of identifying potential interference. To achieve this, analysts can scan for quantifying and qualifying ion transitions for the analyte of interest and calculate their relative ratio. This uses the principle that the analyte fragments in a similar way for patient samples as it does for interference-free samples (e.g. calibrators). Thus, comparison of the ion ratio of a patient sample to that of an interference-free sample can help to identify interference. This feature is now integrated into most software packages.
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Documentation detailing the specific steps for development and validation of quantitative LC-MS/MS assays has been reviewed [105]. Moreover, recent guidance specifically covering LC-MS/MS validation is now available and should help promote the standardisation of method development [106].
With improvements in technology allowing faster scan speeds, analysis of multiple analytes in one assay is now routinely undertaken. However, although multiplexed assays can be advantageous, their application in routine clinical work can prove difficult. Producing fresh calibrators for a singleanalyte assay can itself prove challenging since it is always necessary to ensure patient results do not significantly differ from those previously issued. For multiplexed assays, the likelihood of a problem being encountered during the re-calibration process is significantly increased. Some manufacturers are now beginning to produce metrologically traceable LC-MS/MS kits that include all reagents and consumables required for a given assay. This can help promote standardisation and minimise result variability between laboratories.
In conclusion, the implementation of LC-MS/MS in routine clinical laboratories provides scientists and clinicians with state-of-the-art technology that can produce accurate and precise results. Its application to glucocorticoid analysis is particularly relevant where the non-specific cross-reactivity of commercial immunoassays can potentially compromise patient diagnosis and management. Furthermore, the ability of LC-MS/MS to simultaneously measure several analytes potentiates its clinical utility. With careful attention
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to detail during assay validation, many of the pitfalls associated with this technology can be avoided and robust assays developed.
Acknowledgements The authors would like to thank Dr. Finlay Mackenzie of UK NEQAS for granting permission to use the information provided in Figure 1.
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Figure 1: Method variation for cortisol analysis. This is a recent report from the UK NEQAS serum cortisol EQA scheme. The wide dispersion of results is clearly visible along with a bimodal distribution which is attributed to matrix effects. (The authors wish to thank Dr Finlay Mackenzie of UK NEQAS for granting permission to use this report).
Figure 2: Collision induced dissociation of cortisol. Precursor (A) and product (B) ion scans for a 1 mg/L solution of cortisol infused into a Waters Acquity TQD™ LC-MS/MS system. The m/z 363 peak corresponding to the [M+H]+ cortisol ion is clearly visible in panel A. In panel B the precursor ion has been subjected to collision induced dissociation and the product ion scan has detected the major fragments. The structures are the theoretical fragments that correspond to the accurate mass of the product ions.
Figure 3: A sample serum cortisol chromatogram. Chromatograms taken from a routine cortisol analysis as described by Owen et al [17]. The cortisol concentration was quantified as 15.6 nmol/L using the m/z 363>97 transition (A). The presence of prednisolone in this sample has been detected (C), the reported isobaric interference is apparent in the m/z 363>121 transition (B). Panel D represents the internal standard, cortisol-d4.
Figure 4: An overview of endogenous cortisol metabolism. The metabolites shown can be used to provide a comprehensive overview of glucocorticoid metabolism.
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Figure 5: A sample salivary cortisol chromatogram. A chromatogram taken from the routine analysis of a sample for salivary cortisol and cortisone using the method described by Jones et al [83]. The cortisol concentration was quantified as 1.5 nmol/L (A) whereas cortisone was 8.3 nmol/L (C). Panels B and D corresponds to cortisol-d4 and cortisone-d7 respectively.
Table 1: A summary of the methodologies used to measure serum/plasma endogenous glucocorticoids (* = Limit of Detection).
Table 2: An overview of the methodologies used to measure urine endogenous glucocorticoids (* = Limit of Detection).
Table 3: A summary of the methodologies used to measure salivary endogenous glucocorticoids (* = Limit of Detection).
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Figure
Figure 2
A
B
Figure 3
363 > 97 (cortisol quantifier)
A
363 > 121 (cortisol qualifier)
B
363 > 121 (interference)
361 > 147 (prednisolone)
C
367 > 97 (cortisol-d4)
D
Time (mins)
Figure 4
11bHSD2
11bHSD1
Cortisol
Cortisone 5b-reductase 3a-HSD
Allo-tetrahydrocortisol (5aTHF)
Tetrahydrocortisol (5bTHF)
Tetrahydrocortisone (THE)
Figure 5
363 > 121 (cortisol quantifier)
A
367 > 121 (cortisol-d4)
B
361 > 163 (cortisone quantifier)
C
369 > 169 (cortisone-d7)
D Time (mins)
S0960-0760(16)30144-3 http://dx.doi.org/doi:10.1016/j.jsbmb.2016.05.014 SBMB 4729
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Revised date: Accepted date:
2-9-2015 11-5-2016 12-5-2016
Please cite this article as: James M.Hawley, Brian G.Keevil, Endogenous glucocorticoid analysis by liquid chromatography-tandem mass spectrometry in routine clinical laboratories, Journal of Steroid Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2016.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Endogenous glucocorticoid analysis by liquid chromatography-tandem mass spectrometry in routine clinical laboratories.
James M Hawley 1 and Brian G Keevil 1,2
1. University Hospital South Manchester, Manchester, UK. 2. Manchester Healthcare Academy, Manchester, UK
Corresponding author: James M Hawley Address: Clinical Biochemistry Department, Clinical Sciences Building, University Hospital South Manchester, Southmoor Road, Manchester, M23 9LT. Telephone: 0044 161 291 2136 Fax: 0044 161 291 2927 Email: [email protected]
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Highlights
A review of the clinical applications of LC-MS/MS glucocorticoid analysis.
The major endogenous glucocorticoids are discussed.
Serum, urine and salivary cortisol are comprehensively covered.
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Non-standard abbreviations
3-HSD = 3-hydroxysteroid dehydrogenase 5THF = Allo-tetrahydrocortisol 5THF = Tetrahydrocortisol 11HSD1 = 11 beta hydroxysteroid dehydrogenase type I 11HSD2 = 11 beta hydroxysteroid dehydrogenase type II 11-DOC = 11-Deoxycortisol ACTH = Adrenocorticotropin hormone AME = Apparent mineralocorticoid excess APCI = Atmospheric pressure chemical ionisation APPI – Atmospheric pressure photo ionisation CBG = Cortisol binding globulin CRH = Corticotropin releasing hormone CRM = Certified Reference Material EQA = External quality assessment ESI = Electrospray Ionisation GC-MS = Gas chromatography-mass spectrometry GC-MS/MS = Gas chromatography-tandem mass spectrometry HPA = Hypothalamic – pituitary – adrenal HPLC = High performance liquid chromatography LC-MS/MS = Liquid chromatography-tandem mass spectrometry LC = Liquid chromatography ONDST = Overnight dexamethasone suppression test
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LLE = Liquid-liquid extraction m/z = mass to charge ratio NEQAS = National External Quality Assurance Scheme PFP = Pentafluorophenyl PPT = Protein precipitation RMP = Reference measurement procedure SLE = Supported liquid extraction SPE = Solid phase extraction THE = Tetrahydrocortisone UFC = Urine free cortisol UPLC = Ultra high performance liquid chromatography
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Abstract Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical technique that offers exceptional selectivity and sensitivity. Used optimally, LC-MS/MS provides accurate and precise results for a wide range of analytes at concentrations that are difficult to quantitate with other methodologies. Its implementation into routine clinical biochemistry laboratories has revolutionised our ability to analyse small molecules such as glucocorticoids. Whereas immunoassays can suffer from matrix effects and cross-reactivity due to interactions with structural analogues, the selectivity offered by LC-MS/MS has largely overcome these limitations. As many clinical guidelines are now beginning to acknowledge the importance of the methodology used to provide results, the advantages associated with LCMS/MS are gaining wider recognition. With their integral role in both the diagnosis and management of hypo- and hyperadrenal disorders, coupled with their widespread pharmacological use, the accurate measurement of glucocorticoids is fundamental to effective patient care. Here, we provide an up-to-date review of the LC-MS/MS techniques used to successfully measure endogenous glucocorticoids, particular reference is made to serum, urine and salivary cortisol.
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Introduction Glucocorticoids are steroid hormones that exert their physiological effects through an interaction with complementary glucocorticoid receptors. These receptors are expressed within almost all human cells. Their stimulation initiates a signalling cascade that terminates in the activation of genes that help regulate normal development and control day-to-day homeostasis. The synthesis, metabolism and the pleiotropic actions of glucocorticoids have been extensively reviewed [1, 2, 3]. Similarly, the clinical indications for their measurement have been outlined in international guidelines [4, 5] and recommendations [6].
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique that has transcended research and analytical chemistry laboratories into routine clinical diagnostics [7]. The exquisite sensitivity and specificity LCMS/MS offers is however reliant on the optimisation of several processes.
This review aims to provide the reader with an up-to-date account of LCMS/MS endogenous glucocorticoid measurement. The optimisation of all relevant LC-MS/MS processes including: sample preparation, mobile phase composition, chromatographic separation and MS/MS detection are considered in this review, with particular reference made to serum, urine and salivary cortisol quantification.
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Endogenous glucocorticoids In humans, the major circulating glucocorticoid is cortisol. This can be measured in many biological specimens including: whole blood, plasma, serum, urine, saliva and hair. This variety of sample types serves many different purposes from clinical diagnosis, research studies and athletic drug screening, with each matrix offering its own analytical advantages and challenges. However, in routine clinical biochemistry, only serum, urine and saliva have found prominence with their use is endorsed for the assessment of the hypothalamic-pituitary-adrenal (HPA) axis [4,5,6].
Serum/plasma cortisol Cortisol is one of the best characterised analytes routinely measured in clinical laboratories. Since 1973, the National Institute for Standards and Technology have made available cortisol standard reference material to promote assay standardisation. Subsequently, several reference measurement procedures (RMPs) have been described that utilise this reference material for calibration. These methods have been used to produce higher-order certified reference materials (CRMs) that are metrologically traceable to SI units [8].
The majority of these RMPs have been validated using isotope dilution (ID) gas chromatography-mass spectrometry (GC-MS). However, advances in sample preparation, liquid chromatography and mass spectrometry have
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heralded a second generation of LC-MS/MS reference systems. The first of these was described in 2004 and utilises sequential off-line solid phase extraction (SPE) and liquid-liquid extraction (LLE) for sample preparation prior to LC-MS/MS analysis [9]. More recently, an on-line SPE method has been described [10] and our own group have developed a candidate RMP using supported liquid extraction (SLE) technology for sample clean-up [11].
Although RMPs offer accurate and precise results, they do not represent a timely and cost-effective option for routine analysis. Instead, they are used to assign materials with a target value so they can serve as certified reference materials (CRMs) to calibrate commercial immunoassay kits [12]. However, despite the availability of serum cortisol RMPs and CRMs, there remains a problem with the standardisation of routine commercial immunoassays. External quality assessment (EQA) schemes provide laboratories with a means to review the analytical performance of their method relative to that of other users. Whereas the intra-method variation for a given sample may be relatively small, it can be significantly increased when all methods are accounted for. The report below provides an example of this variation (Figure 1).
[Insert Figure 1 here.]
This longstanding poor performance has been attributed to matrix effects and poor specificity [13, 14]. Consequently, these assays present a potential risk to accurate patient diagnosis and management. This has provided a
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requirement for robust and accurate cortisol methods in routine clinical laboratories. These requirements have been fulfilled by both LC-MS/MS and gas chromatography coupled to either a single quadrupole mass spectrometer (GC-MS) or a triple quadrupole mass spectrometer (GC-MS/MS). However, whereas serum/plasma cortisol analysis by GC-MS and GC-MS/MS has predominantly been limited to RMPs, LC-MS/MS has been favoured for routine clinical analysis. This may be explained by the general requirement to derivatise non-volatile compounds such as cortisol to make them amenable to GC analysis [8]. Despite improving sensitivity, derivatisation can extend sample preparation time and increase total analytical error. Moreover, it has been suggested that the conditions used for derivatisation can hydrolyse conjugates and metabolites to produce the analyte of interest, thereby reducing analytical specificity [15]. Nevertheless, GC-MS and GC-MS/MS still hold an advantage over LC-MS/MS owing to their higher chromatographic resolution, they therefore remain powerful techniques for specialised applications such as urine steroid profiling [16]. Conversely, LC-MS/MS assays do not generally require derivatisation, are typically higher-throughput and now have comparable sensitivity and specificity to GC-MS or GC-MS/MS. Consequently, their application has generally been favoured for routine clinical diagnostics.
Several LC-MS/MS methods have been used to successfully quantify serum cortisol (Table 1). Of those listed, sample volume requirements range from 25 to 1000 µL, with larger volumes generally necessary to improve assay
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sensitivity. Accordingly, many of these assays are designed to quantify a panel of endogenous and exogenous glucocorticoids, many of which are present in significantly lower concentrations than that of cortisol. However, in routine clinical practice, it is notable that serum and plasma are not always of sufficient volume for the multitude of tests that are typically required. This can be especially true of paediatric samples. Hence, for serum/plasma cortisol analysis, small sample volumes are generally preferable.
Sample pre-treatment and extraction is variable across methods (Table 1). The choice of sample preparation may be influenced by a number of factors including: the relative abundance and physiochemical properties of the analyte of interest, sample matrix, potential interferences, the presence of ion suppression or enhancement, the sensitivity required, the time in which the result needs to be issued, operator proficiency and financial considerations. For serum cortisol, successful sample clean-up has been achieved in a number of ways including: protein precipitation (PPT) [17, 18, 19], LLE [20, 21, 22, 23, 24], SPE [25, 26, 27] and also using a combination of these procedures [28, 29, 30].
Collectively, these approaches aim to remove protein and other potential interferences. Deproteinisation reduces the risk of blocking frits, injectors, columns or narrow bore HPLC tubing and helps reduce matrix effects which can have detrimental effects on analysis [31, 32]. In addition to protein, salts and phospholipids can co-elute with the analyte of interest thereby altering the ionisation efficiency. This can lead to in-source competition for ionisation
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resulting in ion suppression or enhancement [31]. This in turn may compromise the accuracy of the final result. Sample deproteinisation is particularly relevant to serum/plasma cortisol analysis. At present, all current clinical guidelines advocate total cortisol measurement [4,5]. Since cortisol circulates as both free (5%) and protein bound (95%) fractions, it is only through liberating cortisol from its binding proteins that allows its total concentration to be reliably quantified.
PPT methods rely on the addition of a precipitant to liberate cortisol from its binding proteins. Several options are available with metal ions [17] and organic solvents [18,19] popular choices. The relative merits and limitations of different forms of PPT have been extensively tested [33]. PPT methods are rapid, amenable to automation, and relatively inexpensive. However, as a consequence of the required sample dilution, assay sensitivity is reduced. Nevertheless, for cortisol alone, this does not appear to jeopardise the utility of the assay since LOQs below current clinical requirements are still achievable, even with sample volumes as small as 25 µL [17].
Conversely, as both LLE and SPE remove more matrix effects and concentrate the sample, both sensitivity and specificity are improved. These extraction techniques have therefore been favoured for multiplexed assays where additional sensitivity and specificity may be required [20, 21, 22, 23, 25, 26, 27, 28, 29, 30]. However, relative to protein precipitation, LLE and in particular SPE, are technically more demanding, require a longer preparation
Page 11 of 66
time and are more expensive. For these reasons they are not always practical for routine clinical work.
Page 12 of 66
Table 1
Analyte(s)
Sample volume (L)
Preparation
Column
Mobile phase (organic/buffer/modifier)
Run time (mins)
Ionisation
LOQ (nmol/L)
Reference
Cortisol
25
PPT (Zinc sulphate / Methanol)
C8
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid
2.6
ESI +
12.5
[17]
Cortisol
250
LLE (Ethyle acetate)
C18
Methanol / 1 mmol/L ammonium chloride / NA
8
ESI -
1.38
[24]
Cortisol, Cortisone
100
LLE (MTBE)
Phenyl
Methanol / NA / NA
3
APPI +
13.76 (Cortisol) 13.87 (Cortisone)
[20]
Cortisol, Cortisone
100
LLE (Ethyle acetate)
C18
Methanol / 5 mmol/L ammonium acetate / NA
Not Stated
ESI +
2.75 (Cortisol), 6.94 (Cortisone)
[23]
Cortisol, Dexamethasone
1000
PPT (Acetonitrile) / SPE (C18)
C18
Methanol / NA / 0.2% formic acid
Not Stated
ESI +
< 20.0 (Cortisol)
[29]
Cortisol, Cortisone, Dexamethasone
200
SPE (Strata X)
Aromax
Acetonitrile / NA / 0.05% formic acid
Not stated
APCI +
0.8 (Cortisol) 0.8 (Cortisone)
[25]
Cortisol, Dexamethasone, Prednisone, Prednisolone
500
SPE (HLB)
C18
Methanol / 5 mmol/L ammonium acetate / NA
6
ESI -
14.85 (Cortisol)
[26]
Cortisol, Cortisone, Prednisolone, Prednisone
50
PPT (Acetonitrile), evaporation, reconstitution
Phenyl
Acetonitrile / NA / 0.1% formic acid
8
ESI +
2.76 (Cortisol), 1.4 (Cortisone)
[18]
Cortisol, Cortisone, 17-OHP, 21-DOC, 11-DOC, Deoxycorticosterone
500
LLE (Methylene chloride)
C18
Methanol / NA / NA
10
ESI +
2.75 (Cortisol),* 13.87 (Cortisone)* 14.4 (11-DOC) *
[21]
Cortisol, Cortisone, Prednisolone, Dexamethasone, 11-DOC,
250
SPE (HLB)
C18
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid
3
ESI +
3.75 (Cortisol), 3.75 (Cortisone), 5.00 (11-DOC)
[27]
Cortisol, Cortisone, Dexamethasone, Prednisolone, Prednisone, Methylprednisolone
500
PPT (Acetonitrile) / LLE (Dichloromethane)
C18
Methanol / 2mmol/L ammonium acetate / 0.1% formic acid
12
ESI +
8.3 (Cortisol), 8.3 (Cortisone)
[28]
Cortisol, Cortisone, Testosterone, Dihydrotestosterone, Progesterone
100
PPT (Methanol)
C18
Methanol / NA / 0.1% formic acid
12
ESI +
5.24 (Cortisol), 0.83 (Cortisone)
[19]
85
LLE (Ethyl acetate:hexane, 80:20 (v/v))
C18
Acetonitrile / NA / 0.1% formic acid
6.1
ESI -
1.95 (Cortisol), 1.58 (Cortisone), 0.098 (11-DOC)
[22]
500
PPT (HCl) / SPE (HLB)
C18
Methanol / 5 mmol/L ammonium acetate / NA
Not stated
ESI -
9.93 (Cortisol)
[30]
Cortisol, Cortisone, 11-DOC, Dexamethasone, Prednisolone, Prednisone, Testosterone, Androstendione, Progesterone Cortisol, Dexamethasone, Methylprednisolone, Prednisone, Prednisolone, Mycophenolic acid, Mycophenolic acid glucuronide
Page 13 of 66
There is marked heterogeneity in mobile phase composition across the methods listed in Table 1. Whereas water is typically used for the aqueous phase, the choice of organic solvent varies between methanol and acetonitrile. As a protic solvent, methanol can promote [M+H]+ ion formation. However, when mixed with water, its viscosity can increase resulting in a high column backpressure. This is generally undesirable and can result in column deterioration or system leaks. Since the advent of UPLC, there has been a trend towards using sub 2 micron particle columns as they can enhance peak resolution. Our experience has found these columns to be especially useful for steroid analysis as they can help separate isobaric interferences [34]. However, columns with < 2 m particles can be susceptible to rapid degradation when subjected to very high backpressures. This can result in deterioration of chromatographic separation and often necessitates that low flow rates are used which consequently increases injection-to-injection time. Acetonitrile offers a useful alternative to methanol as it has a lower viscosity when mixed with water and therefore produces lower backpressures. However, as an aprotic solvent, acetonitrile does not enhance [M+H]+ ion formation, is generally more expensive and, at times, of limited availability.
While several methods do not make use of a buffer [18, 19, 20, 21, 22, 25, 29], others have used ammonium acetate [17, 28, 26, 27, 30, 23] or ammonium chloride [24]. The presence of a buffer can improve the ionisation of cortisol, this may be through adduct formation (e.g. ammonium chloride) or through enhancing in-source ionisation (e.g. ammonium acetate). As a neutral molecule, control of pH has minimal effect on cortisol analysis however,
Page 14 of 66
formic acid has been used to enhance ionisation both in the positive and negative ionisation modes [17, 18, 19, 22, 25, 28, 29, 27]. In the positive ionisation mode, formic acid can provide a rich supply of protons which favour the formation of [M+H]+ ions. Conversely, in the negative ionisation mode, cortisol can form a specific adduct with formic acid as discussed below. In our experience, it is good practice to experiment with different mobile phase compositions to optimise ionisation. In all cases, we have found that LC-MS grade solvents, buffers and modifiers produce the best results. These are typically purer than their HPLC- or reagent-grade equivalents.
By virtue of its hydrophobicity (Log P = 1.43 [35]) and steric properties, cortisol can be chromatographically separated from structurally analogous compounds by traditional alkyl bonded reverse phases. Several C18 columns are now available with differences in end-capping, carbon load and ligand surface area coverage just some of the factors that contribute to the varying performance between columns and manufacturers. Other than C18 columns, a C8 phase has successfully been used [17]. C8 columns rely on similar hydrophobic interactions however, owing to their shorter carbon chain length, analytes are less avidly retained thus permitting shorter run-times. The use of phenyl columns have also been described [18, 20, 25]. This mode of separation relies on pi-pi interactions between aromatic compounds and the stationary phase. Phenyl ligands are therefore capable of exploiting the aromatic ring system present in cortisol to chromatographically separate it from structurally similar compounds. This has reportedly helped separate cortisol from the prednisolone metabolite tetrahydroprednisolone, a potential
Page 15 of 66
isomeric interference [20]. However, in two of the assays developed, it is unlikely that full advantage of the phenyl phase is taking place [18, 25]. In these methods, acetonitrile has been used in the mobile phase. Acetonitrile is known to delocalise the electron clouds that help form pi-pi interactions, therefore suggesting the separations observed involve other retention mechanisms.
Although all assays listed achieve an LOQ below any current clinical requirements, the method described by Ray et al is notably lower than the others (0.80 nmol/L) [25]. This method uses SPE for sample purification and atmospheric pressure chemical ionisation (APCI) to convert cortisol into an ion prior to MS/MS detection. APCI relies on the vaporisation of the column eluent by a heated nebuliser, the remaining molecules (including the analyte of interest) are subsequently ionised by a corona discharge needle. This produces stable ions which then enter the mass spectrometer. APCI is particularly efficient at enhancing the ionisation of compounds that ionise less effectively in solution such as cortisol [36]. This efficiency has contributed to APCI generally being considered a harder form of ionisation than alternatives. Indeed, it has been suggested that APCI can cause an exchange between the deuterated atoms of labelled internal standards with the hydrogen atoms of water therefore compromising accurate quantification [37]. This observation has also been refuted, suggesting instrument-specific differences may effect ionisation [36]. In a novel approach, one cortisol method has used atmospheric pressure photoionisation (APPI) [20]. APPI uses ultraviolet light to irradiate the column eluent which initiates a cascade of reactions resulting
Page 16 of 66
in the formation of stable ions which are channelled into the mass spectrometer. Other than APCI and APPI, the majority of methods have used ESI to ionise cortisol. In ESI, the column eluent enters a stainless steel capillary to which a voltage is applied. A heated gas, usually nitrogen, is passed along the capillary resulting in the nebulisation of the eluent and production of charged droplets. These droplets undergo coulombic fission and are converted into stable ions through desorption and desolvation before entering the mass spectrometer. Relative to APCI and APPI, ESI in the positive ionisation mode does not remove as many matrix effects [37, 38]. However, if careful attention is paid to sample preparation and chromatography, this is not problematic for serum/plasma cortisol analysis since all ESI methods listed in Table 1 achieve sufficient performance characteristics to be clinically useful.
Despite the variation in preparation, separation and ionisation, the transitions used to detect cortisol are similar across methods. To complement this section of the review please refer to Figure 2. This shows a precursor ion scan in the positive ionisation mode of a 1 mg/L methanolic solution of cortisol infused into a Waters Acquity TQD™ LC-MS/MS system under the conditions described by Owen et al [17] (Fig. 2A). Here, the m/z 363 peak corresponding to the [M+H]+ cortisol ion is clearly visible, it is this ion that is scanned for in the first quadrupole of all methods using the positive ionisation mode. In figure 2B we present the product ion scan acquired from applying a collision energy of 28 eV to the precursor ion in the collision cell. In addition,
Page 17 of 66
we have included the corresponding theoretical fragments for the common product ions (Fig. 2B).
The majority of assays listed in Table 1 have favoured the quantification of cortisol using the m/z 363>121 transition [20, 23, 25, 27, 28, 29]. This corresponds to detection of the A-ring fragment following cleavage of the adjoining B-ring bonds [39]. When optimised, the m/z 363>121 is generally the most sensitive transition however, it is not always specific with interference reported from prednisolone and its metabolites [11, 17, 18] (Figure 3). Conversely, the m/z 363>327 [18, 19] and m/z 363>97 [17] transitions have been found to be more selective yet not as sensitive. Whereas the m/z 363>327 transition corresponds to cleavage of two hydroxyl groups, the m/z 363>97 transition represents cleavage and subsequent rearrangement on the A-ring fragment [40, 41]. In addition, Ray et al have also used m/z 363>267 as a secondary transition.
[Insert Figure 2 here]
[Insert figure 3 here]
Whereas ESI in the positive ionisation mode is favoured for cortisol analysis, four of the methods have used ESI in the negative ionisation mode [22, 24, 26, 30]. Of these, two have exploited of the ability of cortisol to form an adduct
Page 18 of 66
in this mode. Huang et al have used ammonium chloride to form a [M+35]- precursor ion and subsequently scanned for the m/z 397>35 product ion following fragmentation of cortisol and the chloride ion in the collision cell [24]. This allows for a very sensitive method however; it is notable that scanning for product ions in the lower range of the mass scale is generally not recommended as specificity can be compromised. The method described by Methlie et al has instead monitored the formic acid adduct [M+46] - and the corresponding m/z 407>297 transition following collision induced dissociation [22]. Conversely, two methods do not rely on adduct formation and instead scan for the native [M-H]- ion which fragments to [M-H-CH2O]-, this corresponds to m/z 361>331 [26, 30]. Relative to ESI in the positive ionisation mode, detection of cortisol in the negative ionisation mode has been reported to improve signal-to-noise [26, 30].
Interestingly, four of the methods summarised have used structural analogues as an internal standard including: flumethasone [26, 30], prednisolone-d6 [28] and 6-methylprednsiolone [24]. To ensure the internal standard provides true compensation for variability in ionisation, its retention time should be approximately equal to that of the analyte of interest. Therefore, given their widespread availability, it is highly recommended that isotopically labelled internal standards are used in preference to structural analogues for assay validation.
In general, short run times are desirable for a prompt result turnaround. The assay developed by Owen et al has been specifically designed for
Page 19 of 66
implementation into a routine clinical laboratory [17]. The other assays listed in Table 1 are mainly multiplexed and designed to ensure the adequate separation of all compounds scanned for. As such, they require longer run times.
One application where LC-MS/MS serum cortisol quantification has been found to be increasingly important is in patients prescribed metyrapone. This 11-hydroxlase inhibitor is used to block cortisol synthesis in patients with Cushing’s syndrome. In addition, it is used in some countries to help diagnose adrenal insufficiency. Serum cortisol measurement by routine immunoassays has been found to be prone to interferences in samples collected from patients prescribed metyrapone [42, 43]. Inhibition of 11- -hydroxlase can cause gross elevations in cortisol precursors (e.g. 11–deoxycortisol) that can interfere with immunoassay cortisol quantification therefore potentially masking hypoadrenalism [43, 44]. As metyrapone dosing regimens may be titrated against cortisol concentrations this has important ramifications on patient care. This fact has been recognised by the Endocrine Society in their recent clinical practice guideline concerning Cushing’s Syndrome treatment [44]. In this document, LC-MS/MS is recommended for serum and urine cortisol quantification in patients prescribed metyrapone as it circumvents the problems associated with immunoassays. McWhinney et al also suggest that when metyrapone is administered as part of a dynamic function test for the diagnosis of adrenal insufficiency, concomitant measurement of 11deoxycortisol along with cortisol can aid result interpretation [27].
Page 20 of 66
Urine free cortisol and its metabolites Urine free cortisol (UFC) is advocated for the investigation of hypercortisolism because it represents the non-protein bound cortisol fraction that is freely filtered at the glomerulus [4]. Whereas immunoassays suffer from interference due to cross reactivity with cortisol A-ring metabolites and other steroids, the selectivity and sensitivity of LC-MS/MS overcomes this to provide accurate results [45]. Consequently, many urinary free cortisol LC-MS/MS methods have been published (Table 2).
Urine cortisol measurements are only recommended following a 24h collection period [4], hence obtaining sufficient sample volume is rarely problematic. This is reflected by the relatively larger volumes that are used in urine assays compared to that of serum (Table 2).
Two of the methods listed make use of the simplest form of sample preparation namely ‘dilute-and-shoot’ [46, 47]. This can significantly reduce both preparation time and inter-analyst variation. However, this is at the expense of introducing relatively dirty samples into the LC-MS/MS system. This can be detrimental as the risk of blocking tubing or frits increases, column lifetime can reduce, and the mass spectrometer itself may require additional services as salts can deposit within the system. Although more comprehensive sample clean-up is recommended, analysts wishing to explore dilute-and-shoot should at least centrifuge the specimen prior to sampling to ensure particulates do not enter the LC-MS/MS system. In an evolution of dilute-and-shoot, Kushnir et al al have used a 2D chromatography system
Page 21 of 66
with an online C18 guard cartridge to trap cortisol [36]. In this method, the sample is first injected onto a guard column where it is trapped and subjected to an organic wash. This wash is sufficiently organic to remove hydrophilic interferences to waste without disrupting the interaction between the analyte of interest and the guard column phase. After the sample is suitably purified, the flow path is switched and the guard column eluent is diverted to the analytical column. The organic composition of the mobile phase is then increased to liberate the analyte of interest from the guard column onto the analytical column. This allows further sample clean-up in the form of traditional reverse-phase chromatography prior to MS/MS detection.
Page 22 of 66
Table 2 Analyte(s)
Sample volume (L)
Preparation
Column
Mobile phase (organic/buffer/modifier)
Run time (mins)
Ionisation
LOQ (nmol/L)
Reference
Cortisol
500
dilution (1:1 water)
C18
Methanol / NA / 0.003% trifluoroacetic acid
3
APCI +
13.8
[46]
Cortisol
500
Online C18 cartridge
C18
Methanol / 5 mmol/L ammonium formate / NA
8
APCI +
5.5
[36]
5
ESI +
21.5
[48]
4.5
ESI +
2
[38]
8
ESI -
1.38 6. 0 (Cortisol), 6.0 (Cortisone)
[24]
Cortisol
100
PPT (trichloroacetic acid)
C18
Cortisol
1000
SPE (HLB)
C18
Cortisol
250
LLE (ethyl acetate)
C18
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid Methanol / 2mmol/L ammonium acetate / 0.1% formic acid Methanol / 1 mmol/L ammonium chloride / NA
Cortisol, Cortisone
500
LLE (methylene chloride)
C18
Methanol / NA / NA
3
ESI +
Cortisol, Cortisone, 6-sulfatoxymelatonin
250 (minimum)
dilution (1:1 water)
C18
Acetonitrile / NA / 0.1 % formic acid
6
ESI +
3.8 (Cortisol) * 4.7 (Cortisone) *
[47]
Cortisol, Cortisone
100
Turboflow
C18
Methanol / NA / NA
9.5
APCI +
5.5 (Cortisol) 2.8 (Cortisone)
[54]
Cortisol
300
SPE (C8 Column)
C18 monolithic
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid
4.5
ESI +
5
[50]
Cortisol, Cortisone
1000
SPE (MAX)
C8
Methanol / 20 mmol/L ammonium formate / NA
10
ESI +
0.4 *
[51]
Cortisol, Cortisone, Tetrahydrocortisol, Allotetrahydrocortisol, Tetrahydrocrotisone Cortisol, Cortisone, Tetrahydrocortisol, Allotetrahydrocortisol, Tetrahydrocrotisone Cortisol, Cortisone, Tetrahydrocortisol, Allotetrahydrocortisol, Tetrahydrocrotisone
1000
SPE (HLB)
C18
Methanol / NA / 0.1% formic acid
18
ESI +
2000
SPE (HLB) / dilution (1:1 water)
Biphenyl
Methanol / NA / 0.1% formic acid
19.5
ESI -
480
PPT (acetonitrile)
C18
25
ESI -
Acetonitrile / 5 mmol/L ammonium acetate / 0.1% formic acid
Page 23 of 66
0.3 (Cortisol) 0.3 (Cortisone) 2.7 (Tetrahydrocortisol) 2.7 (Allo-tetrahydrocortisol) 2.3 (Tetrahydrocortisone) 8.3 (Cortisol) 1.7 (Cortisone) 0.8 (Tetrahydrocortisol) 0.8 (Allo-tetrahydrocortisol) 1.6 (Tetrahydrocortisone) 0.14 (Cortisol) 0.14 (Cortisone) (Tetrahydrocortisol) (Allo-tetrahydrocortisol) (Tetrahydrocortisone)
[37]
[52]
[53]
[49]
Alternative approaches to sample preparation have involved protein precipitation with trichloroacetic acid [48] or acetonitrile [49], LLE using ethyl acetate [24] or methylene chloride [37] and SPE [45, 50, 51, 52, 53]. With its enhanced purification providing improved sensitivity and specificity, SPE is the favoured approach. The advantages SPE confers are highlighted by the lower limits of quantification achieved using this technique relative to others. Although few authors disclose any preliminary work that influenced their choice of SPE phase, Fong et al evaluated their method using both a mixed mode anion exchange (MAX) phase and a mixed mode cation exchange (MCX) phase, superior recovery and imprecision were observed using the MAX cartridges [51]. In a novel approach, Sánchez-Guijo et al have introduced samples directly into a Turboflow™ chromatography system [54]. This uses turbulent solvent flow in a short chromatography column packed with porous particles to separate smaller molecules from larger ones (e.g. proteins) in complex biological matrices. Whereas the small molecules interact with particle pores, larger ones are size-excluded and discarded. Once separated, an analytical column can be used for improving separation of the smaller molecules prior to MS/MS analysis. The use of the Turboflow™ dramatically reduces bench-time preparation and inter-operator variability.
The majority of methods listed in Table 2 achieve chromatographic separation of cortisol using the hydrophobic interaction of C8 and C18 columns. However, in two original approaches, one method makes use of a monolithic phase [50] whereas another utilises a biphenyl column [53]. Monolithic phases rely on a silica based porous rod system to achieve chromatographic
Page 24 of 66
separation. The stability of these porous rods allow higher flow rates (up to 1 mL/min) without the consequential rise in pressure that is observed with particle based chromatography. This permits the development of efficient and rapid methods. Conversely, the biphenyl phase, like its phenyl counterpart, relies on the pi-pi interaction between hydrophobic aromatic compounds and the biphenyl ligand, this can produce exceptional separation of structurally similar compounds such as the stereoisomers tetra-hydrocortisol and allotetra-hydrocortisol [53].
Although most assays have used an isotopically labelled internal standard, one method has used 6--methylprednisolone to compensate for variation in ionisation [47]. Interestingly, another method evaluated methylprednisolone as an alternative for cortisol-d4 [53]. The results demonstrated that the structural analogue was 10-15% less accurate than the isotopically labelled internal standard for the quantification of cortisol and its metabolites.
Whereas ESI in the positive ionisation mode is the most common approach, three methods have used ESI in the negative ionisation mode [24, 49, 53]. Under these conditions, Cuzzola et al specify that the formic acid adduct [M+HCOO]- consistently fragments to [M-H-CH2O]- for all analytes providing high sensitivity and specificity [53]. In contrast, three methods have utilised APCI in the positive ionisation mode, this reportedly increased signal-to-noise by 2-3 fold compared to ESI [36, 46, 54]. Despite the differences used to achieve ionisation, all methods have determined an LOQ within the desirable clinical range for routine UFC measurements. The differences observed for
Page 25 of 66
ionisation may be directly related to the buffers and modifiers used, or the insource ionisation which can vary between manufacturers
Aside from hypercortisolism, quantification of urine cortisol and its metabolites can also be effective in the diagnosis of other pathologies related to disordered steroid production or metabolism. Several enzymes are involved in modulating the availability, metabolism and clearance of cortisol (Figure 4) [55]. Hepatic and adipose tissue both express the enzyme 11-hydroxysteroid dehydrogenase type I (11HSD1) which converts the inactive cortisone to the active cortisol. This reaction can also be reversed in the presence of 11hydroxysteroid dehydrogenase type II (11HSD2) which is expressed in renal and colon cells. Furthermore, the action of the hepatic A-ring reductases (5 and 5reductase), in conjunction with 3-hydroxysteroid dehydrogenase (3-HSD), inactivate cortisol to the tetrahydrometabolites: allotetrahydrocortisol (5THF), tetrahydrocortisol (5THF), and tetrahydrocortisone (THE). Thus, as conjugation is predominantly a hepatic process, the ratio of cortisol and cortisone tetrahydrometabolites (i.e. 5THF + 5THF / THE) provides an index of hepatic 11HSD1 activity. Similarly, the free cortisol:cortisone ratio provides information on the activity of 11HSD2. In addition, the ratio of 5THF / 5THF reflects the relative activities of global 5 and 5reductase activity [56, 57].
[Insert Figure 4 here]
Page 26 of 66
Much of the early work measuring these metabolites was undertaken using GC-MS with the syndrome of apparent mineralocorticoid excess (AME) one of the first conditions identified [58]. AME is characterised by a congenital defect in 11HSD2 which prevents the oxidation of cortisol to cortisone. Without this protective mechanism, cortisol can accumulate and stimulate the mineralocorticoid receptor (MR). Interestingly, AME can also be acquired through inhibition of 11HSD2 secondary to carbenoxolone therapy or excessive consumption of glycyrrhizinic acid (commonly found in liquorice). Unregulated MR stimulation results in sodium and water retention consequently leading to hypertension. Two congenital forms of AME have been described, both of which can be determined by virtue of an elevated urine free cortisol to cortisone ratio [59]. However, Type II is distinguishable from Type I as A-ring reduction is the major deficiency [60, 61].
In addition to AME, GC-MS has helped to identify glucocorticoid metabolic imblances in patients with glucocorticoid suppressible aldosteronism and adrenal adenomas [62, 63], females with polycystic ovarian syndrome [64], obesity [65], and congenital adrenal hyperplasia secondary to 21-hydroxylase deficiency [66]. Furthermore, a debate has recently emerged in the literature suggesting that the cross-reactivity observed with cortisol metabolites in immunoassays may actually increase their diagnostic sensitivity for Cushing’s Syndrome [67]. Therefore, to further increase the diagnostic sensitivity of LCMS/MS it may be necessary to measure cortisol and its metabolites to provide a complete assessment of hypercortisolism. Collectively, this evidence has
Page 27 of 66
provided an impetus for the development of LC-MS/MS assays capable of measuring cortisol and its metabolites.
Consequently, several of the assays listed in Table 2 have been developed to measure cortisone concomitantly with cortisol [37, 47, 49, 51, 52, 53, 54]. In comparison to cortisol, cortisone differs by the presence of a C=O group in position 11. Taylor et al suggest that the presence of this bond may be responsible for the different ionisation pattern observed with cleavage of the C-ring (as opposed to the B-ring with cortisol) producing the most abundant fragment m/z 361 > 163 when analysed in the positive ionisation mode [37]. Interestingly, as prednisolone and cortisone share the same molecular weight (360.44 Da) crosstalk has also been reported in their analysis [22, 28]. This can be prevented by ensuring adequate chromatographic separation of the two compounds.
In normal adults, approximately twice as much cortisone is excreted compared to cortisol, as such sensitivity has not proved problematic. Hence, it has been suggested that the the cortisol:cortisone ratio can add value to result interpretation and the evaluation of 11HSD2 activity [68].
More recently, several LC-MS/MS assays have been designed to provide a more complete picture of cortisol metabolism through measurement of cortisol, cortisone and their tetrahydrometabolites: 5THF, 5THF and THE. One of the earliest methods published quantified only the tetrahydrometabolites and required a derivatisation procedure that increased
Page 28 of 66
sensitivity 15-80-fold [69]. Campino et al have since published a method using an F5 column that did not allow for chromatographic separation of the stereoisomers 5THF and 5THF and therefore provided limited information regarding the 5 and 5reductase activity [70]. F5 columns act as a lewis acid due to the presence of 5 electronegative fluorine groups. This ensures the phenyl ring is electron deficient which can help separate closely related ring systems such as those present in steroids. However, care is still required to ensure the gradient encourages separation of closely related compounds such as stereoisomers. More recently a series of methods have been published that achieve separation of all compounds including the 5THF and 5THF [49, 52, 53, 71]. Interestingly, two of the methods have favoured ESI in the negative ionisation mode owing to its enhanced signal-to-noise relative to ESI in the positive ionisation mode [53] However, this observation may be instrument-specific and it is always worth experimenting in both modes. Importantly, the cycle times injection-to-injection for these three assays range from 18 – 25 minutes [49, 52, 53, 71], run times this long are not always conducive with routine, high-throughput laboratories. Another potential problem with these assays is they are unable to truly compensate for variation in ionisation due to a lack of availabile isotopically labelled internal standards. Taking the method of Zhai et al as an example, here the structural analogue methylprednisolone elutes after approximately 6 minutes, the analytes of interest subsequently elute between 8 and 18 minutes [49]. Thus, it is apparent that there is a significant period where no allowance is made for variation in ionisation efficiency. In an original approach, Marcos et al have
Page 29 of 66
used a C18 column to achieve excellent separation of a panel of 67 corticosteroids including many additional glucocorticoid metabolites [71].
Salivary cortisol By virtue of its lipophilic nature, serum free cortisol can passively diffuse into saliva via the acinar cells in a process that is independent of salivary flow rate [72]. Therefore, like UFC, salivary cortisol quantification provides an index of serum free cortisol concentration [73]. The use of saliva for cortisol measurement has several advantages over both urine and serum: as a matrix itself, saliva contains relatively fewer interfering compounds and in that regard presents less of an analytical challenge. In addition, its non-invasive, costeffective collection is appealing to both patients and clinicians. Consequently, advances in sampling techniques coupled with a strong evidence base have propelled salivary cortisol from a novel assay confined to endocrine and psychological research into routine clinical practice. Indeed, it is now recommended as a first-line diagnostic test in the investigation of Cushing’s Syndrome [4]. Its transition into clinical laboratories has in part been driven by LC-MS/MS with accurate quantification of low salivary cortisol concentrations now possible. Originally measured by RIA and immunoassay, salivary cortisol has been reported to suffer from non-specific cross-reactivity with endogenous and exogenous glucocorticoids. In addition, poor standardisation between assays has contributed to assay-specific variation meaning results are not directly comparable [74, 75]. This problem is compounded by the expression of 11HSD2 in the parotid glands which ensures the cortisone:cortisol ratio is significantly higher than in serum.
Page 30 of 66
The structural similarity of cortisone to cortisol has proved difficult for immunoassays to differentiate between the two. These limitations have largely been circumvented by the application of LC-MS/MS. Table 3 provides a summary of recently published LC-MS/MS salivary cortisol assays.
Page 31 of 66
Table 3
Analyte(s)
Sample Volume (L)
Preparation
Column
Mobile Phase (organic/buffer/modifier)
Run time (mins)
Ionisation
LOQ (nmol/L)
Reference
Cortisol
100
LLE (dichloromethane)
C18
Methanol / NA / NA
10
ESI -
0.07
[78]
Cortisol
100 / 200
PPT (heat denaturation) /online SPME (Supel Q PLOT capillary)
C8
Methanol / NA / 1% acetic acid
5
ESI +
0.01*
[80]
Cortisol
250
PPT (acetonitrile / acetic acid)
C8
Methanol / NA / 0.5% acetic acid,
5
ESI +
1.4
[79]
Cortisol, cortisone
50
online SPE (C18)
C18 monolithic
Methanol, 0.1% formic acid
4
ESI +
0.75 (Cortisol) 0.75 (Cortisone)
[83]
Cortisol, cortisone
500
PPT / SPE (strata X) / derivitisation
C18 monolithic
Methanol, 500 mmol/L ammonium formate / NA
Not stated
ESI +
0.66 (Cortisol) 2.77 (Cortisone)
[76]
Cortisol, Cortisone
100
online SPE (C8)
C18
Methanol / 2 mmol/L ammonium acetate /0.1% formic acid
5
ESI +
0.39 (Cortisol) 0.78 (Cortisone)
[81]
Cortisol
200 / 100
online SPE (C8)
C18
Methanol / 2 mmol/L ammonium acetate / 0.1% formic acid
4
ESI +
2
[82]
Cortisol, Cortisone, Melantonin
50
Turboflow
C18
Methanol, 1.5 mmol/L ammonium formate / 0.1% formic Acid
13
ESI +
0.55 (Cortisol), 5.5 (Cortisone)
[84]
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These methods share a common requirement for small sample volumes ranging from 50-250 L however, one method requires 500 µL [76]. Generally, saliva is not a difficult specimen to obtain, with the availability of dedicated collection vessels promoting standardised collection procedures. Nevertheless, despite following these procedures, salivary yields can be poor in paediatric populations and acutely unwell patients. For this reason, small sample volumes remain desirable for routine analysis.
Salivary cortisol quantification is used to assess the nadir of an individuals diurnal rhythm. Consequently, the cortisol concentration in normal subjects at this time can challenge the functional sensitivity of immunoassays. LC-MS/MS analysis is better suited to this task because it is 10-100 fold more sensitive than RIA and confers greater specificity [77]. To achieve this, LC-MS/MS salivary cortisol methods generally benefit from a sample preparation that purifies and concentrates cortisol prior to analysis. This has been accomplished by various techniques including LLE using dichloromethane [78] and protein precipitation with evaporation of the supernatant prior to reconstitution [79]. Heat denaturation has also been combined with an online solid phase micro extraction (SPME) technique that consists of a 2D chromatography system that loads the sample onto a fused silica capillary [80]. In a similar approach the use of off-line [76] or on-line [81, 82, 83] SPE has been used for sample purification. When performed off-line, SPE can take a relatively long time to prepare with the process heavily reliant on operator proficiency. Conversely, online SPE reduces inter-operator variability and ensures maximum recovery of the analyte as loss is minimal compared to the
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offline evaporation of an eluate. Online SPE using cartridges has advantages over the use of a guard column as the washes can be performed separately to the main flow path hence solvents other than those used in the mobile phase can be utilised for sample clean up [83]. Another approach to online sample clean-up has used Turboflow™ technology to purify the sample prior to introduction to the LC-MS/MS, this requires little sample pre-treatment ensuring minimal inter-operator variation [84].
Two assays have been validated using a monolithic C18 phase [76, 83]. As discussed above, the application of a monolithic phase as opposed to a traditional silica particle based column can facilitate the development of rapid and efficient methods such as that described by Jones et al which achieves separation of cortisol and cortisone within 4 minutes (Figure 5) [83].
[Insert Figure 5 here]
Sensitivity can be crucial in salivary cortisol methods, as such the majority of assays listed in Table 3 use ESI in the positive ionisation mode to monitor the m/z 363>121 transition [81, 82, 83, 84]. Conversely, Jonsson et al have reported observing interference in the m/z 363>121 transition and have instead opted to use m/z 363>309 [79]. Vieira et al use hydroxylamine for derivatisation and scan for the corresponding m/z 393>136. Another method has analysed cortisol in the negative ionisation mode using the m/z 361>331 transition [78]. Finally, one method uses just a single quadrupole to scan for the [M+H]+ ion corresponding to m/z 363 [80]. The use of single quadrupole
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mass spectrometers is generally not recommended, specificity is considerably reduced and therefore the chance of detecting an interfering compound is increased.
Salivary cortisone and cortisol can be measured concurrently [76, 81, 83, 84]. It has been suggested that this can facilitate result interpretation [81]. The non-invasive nature of salivary collection lends itself to the assessment of hydrocortisone replacement therapy whereby day-curves can be produced to ensure patients are provided with optimal steroid replacement. Measuring cortisone and cortisol concomitantly can demonstrate contamination from oral hydrocortisone shown by an unexpectedly high cortisol:cortisone ratio [81]. Perogamvros et al have proposed that the salivary free cortisone concentration may more accurately reflect the concentration of free serum cortisol [85].
Although much work has focused on the application of salivary cortisol in the diagnosis of Cushing’s syndrome, there is evidence to suggest it can also be used in the diagnosis of adrenal insufficiency [81, 86, 87]
Future directions Over the past decade there has been an exponential rise in the number of LCMS/MS glucocorticoid publications. This trend does not appear to be declining with advances in sample preparation, chromatography, and mass
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spectrometry providing scientists with the instrumentation required to improve existing techniques. This, coupled with increasing clinical knowledge of glucocorticoid metabolism, is providing a solid platform to continue advancing the field.
For many scientists and clinicians with an interest in glucocorticoid analysis, the development of robust serum/plasma free cortisol assays remains an important goal. Free hormone quantitation is analytically challenging as analysis itself can disturb the equilibrium between the free and protein bound fractions bringing into question the integrity of the final result. Although free serum cortisol assays have been described, they typically require extensive sample preparation such as ultrafiltration [24, 88] or equilibrium dialysis [89]. These techniques are not conducive to high-throughput laboratories and it is possible that advances in sample preparation or LC-MS/MS methodology could advance free hormone analysis into a routine application. This would require extensive clinical studies to support its utility.
The case for LC/MS/MS measurement of urine cortisol, cortisone and their metabolites has been outlined above. Although initial assays have been described, their cycle times are relatively long when compared to most routine LC-MS/MS methods [49, 52, 53]. Future work is required to develop these assays for routine use. In addition, there is a general requirement for manufactures to provide isotopically labelled internal standards for the glucocorticoid metabolites.
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Another potentially exciting development in the field of LC-MS/MS glucocorticoid analysis is the quantification of hair total cortisol. As cortisol has been found to deposit in the hair shaft, it is postulated that hair cortisol analysis may allow clinicians to assess chronic exposure to cortisol to aid in the diagnosis of Cushing’s Syndrome, monitor hydrocortisone replacement therapy and to assess pathologies associated with chronic stress [90]. Despite the growing evidence base [91, 92, 93, 94], the utility of hair cortisol measurements has yet to be recognised in any clinical guidelines. One possible reason for this is the pre-analytical factors involved with hair collection [95]. Further work is required to fully assess the clinical utility of glucocorticoid hair measurements.
It is also important to appreciate that unlike cortisol, no RMPs currently exist for any other endogenous glucocorticoids or their metabolites. For this reason, metrological traceability cannot be guaranteed and assay standardisation may in future be identified as a problem. Thus, there remains a requirement for RMPs to be developed that are sufficiently accurate and precise they can be used to produce CRMs that will promote assay standardisation.
One of the major advantages LC-MS/MS holds over immunoassay is the ability for laboratories to develop novel assays relatively quickly. With the introduction of new equipment and the recognition of new endogenous metabolites opportunities will continue to be presented for novel assay development.
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Historically, one factor that has limited the wider uptake of LC-MS/MS is the relative expense of the equipment compared to other analysers. As the number of manufacturers has increased and technology has advanced, entrylevel instruments have gradually become more affordable. This has helped a greater number of laboratories adopt LC-MS/MS technology. However, their cost and the expertise required to successfully operate them remain an issue, especially in the developing world. To circumvent this, there is a general requirement for manufacturers to develop equipment that can integrate into routine, high-throughput, automated chemistry analysers that will require minimal operator input.
Summary The field of glucocorticoid quantitation is being propelled by LC-MS/MS. With its adoption by routine clinical chemistry laboratories, there has been increasing awareness of the advantages this powerful technology offers over existing methodologies. This recognition has now moved beyond the laboratory with many clinicians and research scientists cognisant of the advantages LC-MS/MS offers for patient diagnosis and management as well as for high impact clinical research.
Despite its growing reputation, there have been general concerns raised regarding LC-MS/MS analysis. Many of these centre on the variability observed across methods that are designed to measure the same analyte [96, 97, 98, 99]. This concern has been justified in this review. Although many methods rely on established validation criteria (e.g. FDA IVD guidelines [100])
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there is widespread variation to the approaches used with many of these, especially the older ones, reliant on inadequate validation.
Several of the methods reviewed have used structural analogues as opposed to a stable, isotopically labelled internal standard. This can be problematic if the internal standard and analyte of interest do not co-elute. To compensate for variation in ionisation efficiency, it is essential that the physiochemical properties of the internal standard resemble those of the analyte of interest as closely as possible. Although isotopically labelled internal standards are recommended over structural analogues, problems can still be encountered. Duxbury et al have shown that when using cortisol-d2 as an internal standard to measure cortisol, the naturally occurring isotope cortisol-365 can falsely increase the internal standard concentration therefore reducing the relative response for cortisol [101]. This can largely be avoided by selecting internal standards with more than 2 substituted deuteriums. Davison et al have also reported that the location of the deuterium labels can be crucial as some sites will back-exchange with hydrogen atoms thereby causing a reduction in internal standard area and an increase the relative response of the analyte [102]. Many of the concerns surrounding internal standards have been addressed with the introduction of superior C13 based compounds [38].
When clinical samples are to be measured, the assay should be validated using samples taken using blood collection tubes from several manufacturers and using different anticoagulants. These could all effect how well the analyte of interest ionises because of differing ionisation efficiency profiles. Matrix
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effects can be evaluated using infusion experiments to qualitatively test for ion suppression [31] and also quantitatively by comparing the recovery of spiked analyte from aqueous and plasma samples [103]. It is also possible to monitor interferences directly using targeted analysis of individual phospholipids [104].
Another common problem is the investigation of analytical interference. Method validation typically includes analysis of a panel of endogenous and exogenous compounds structurally analogous to the analyte of interest. The potential interferent is normally added in excess to a solution and injected into the LC-MS/MS system, any interference is deemed significant if a signal greater than the LOQ of the assay is detected in any of the m/z transitions scanned for. Although sufficient for endogenous compounds, this approach is not comprehensive enough for exogenous compounds since it is often parent compound metabolites that manifest as an interferent. For example, although prednisolone itself can be separated from cortisol, its metabolites have been reported to interfere in LC-MS/MS analysis [11,17, 18]. As it is not always possible to investigate drug metabolites, it is best practice to develop a method capable of identifying potential interference. To achieve this, analysts can scan for quantifying and qualifying ion transitions for the analyte of interest and calculate their relative ratio. This uses the principle that the analyte fragments in a similar way for patient samples as it does for interference-free samples (e.g. calibrators). Thus, comparison of the ion ratio of a patient sample to that of an interference-free sample can help to identify interference. This feature is now integrated into most software packages.
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Documentation detailing the specific steps for development and validation of quantitative LC-MS/MS assays has been reviewed [105]. Moreover, recent guidance specifically covering LC-MS/MS validation is now available and should help promote the standardisation of method development [106].
With improvements in technology allowing faster scan speeds, analysis of multiple analytes in one assay is now routinely undertaken. However, although multiplexed assays can be advantageous, their application in routine clinical work can prove difficult. Producing fresh calibrators for a singleanalyte assay can itself prove challenging since it is always necessary to ensure patient results do not significantly differ from those previously issued. For multiplexed assays, the likelihood of a problem being encountered during the re-calibration process is significantly increased. Some manufacturers are now beginning to produce metrologically traceable LC-MS/MS kits that include all reagents and consumables required for a given assay. This can help promote standardisation and minimise result variability between laboratories.
In conclusion, the implementation of LC-MS/MS in routine clinical laboratories provides scientists and clinicians with state-of-the-art technology that can produce accurate and precise results. Its application to glucocorticoid analysis is particularly relevant where the non-specific cross-reactivity of commercial immunoassays can potentially compromise patient diagnosis and management. Furthermore, the ability of LC-MS/MS to simultaneously measure several analytes potentiates its clinical utility. With careful attention
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to detail during assay validation, many of the pitfalls associated with this technology can be avoided and robust assays developed.
Acknowledgements The authors would like to thank Dr. Finlay Mackenzie of UK NEQAS for granting permission to use the information provided in Figure 1.
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Figure 1: Method variation for cortisol analysis. This is a recent report from the UK NEQAS serum cortisol EQA scheme. The wide dispersion of results is clearly visible along with a bimodal distribution which is attributed to matrix effects. (The authors wish to thank Dr Finlay Mackenzie of UK NEQAS for granting permission to use this report).
Figure 2: Collision induced dissociation of cortisol. Precursor (A) and product (B) ion scans for a 1 mg/L solution of cortisol infused into a Waters Acquity TQD™ LC-MS/MS system. The m/z 363 peak corresponding to the [M+H]+ cortisol ion is clearly visible in panel A. In panel B the precursor ion has been subjected to collision induced dissociation and the product ion scan has detected the major fragments. The structures are the theoretical fragments that correspond to the accurate mass of the product ions.
Figure 3: A sample serum cortisol chromatogram. Chromatograms taken from a routine cortisol analysis as described by Owen et al [17]. The cortisol concentration was quantified as 15.6 nmol/L using the m/z 363>97 transition (A). The presence of prednisolone in this sample has been detected (C), the reported isobaric interference is apparent in the m/z 363>121 transition (B). Panel D represents the internal standard, cortisol-d4.
Figure 4: An overview of endogenous cortisol metabolism. The metabolites shown can be used to provide a comprehensive overview of glucocorticoid metabolism.
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Figure 5: A sample salivary cortisol chromatogram. A chromatogram taken from the routine analysis of a sample for salivary cortisol and cortisone using the method described by Jones et al [83]. The cortisol concentration was quantified as 1.5 nmol/L (A) whereas cortisone was 8.3 nmol/L (C). Panels B and D corresponds to cortisol-d4 and cortisone-d7 respectively.
Table 1: A summary of the methodologies used to measure serum/plasma endogenous glucocorticoids (* = Limit of Detection).
Table 2: An overview of the methodologies used to measure urine endogenous glucocorticoids (* = Limit of Detection).
Table 3: A summary of the methodologies used to measure salivary endogenous glucocorticoids (* = Limit of Detection).
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Figure
Figure 2
A
B
Figure 3
363 > 97 (cortisol quantifier)
A
363 > 121 (cortisol qualifier)
B
363 > 121 (interference)
361 > 147 (prednisolone)
C
367 > 97 (cortisol-d4)
D
Time (mins)
Figure 4
11bHSD2
11bHSD1
Cortisol
Cortisone 5b-reductase 3a-HSD
Allo-tetrahydrocortisol (5aTHF)
Tetrahydrocortisol (5bTHF)
Tetrahydrocortisone (THE)
Figure 5
363 > 121 (cortisol quantifier)
A
367 > 121 (cortisol-d4)
B
361 > 163 (cortisone quantifier)
C
369 > 169 (cortisone-d7)
D Time (mins)