Principles, current applications, and future perspectives of liquid chromatography-mass spectrometry in clinical chemistry

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Principles, current applications, and future perspectives of liquid chromatography-mass spectrometry in clinical chemistry

22

Matteo Ludovici*, Cristiano Ialongo†, Emanuela Camera* San Gallicano Dermatologic Institute (IRCCS), Rome, Italy* Sapienza University of Rome, Rome, Italy†

­C HAPTER OUTLINE 22.1 Introduction..................................................................................................... 727 22.2  Sample Preparation in Clinical LC-MS............................................................... 728 22.3  LC-MS Technologies in Clinical Chemistry......................................................... 730 22.3.1  Liquid Chromatography................................................................ 730 22.3.2  LC-MS Interfaces........................................................................ 733 22.3.3  Mass Spectrometers.................................................................... 734 22.3.4  Quadrupole Analyzers.................................................................. 735 22.3.5  Accurate Mass Analyzers.............................................................. 735 22.4 Applications.................................................................................................... 736 22.4.1  Fat-Soluble Vitamins................................................................... 736 22.4.2  Polyunsaturated Fatty Acids (And Their Metabolites)...................... 739 22.4.3  Endogenous Glucocorticoids........................................................ 741 22.5  Future Perspectives.......................................................................................... 743 22.6 Conclusions..................................................................................................... 744 References............................................................................................................... 744

22.1 ­INTRODUCTION Clinical chemistry is a multifaceted discipline that combines chemistry, biochemistry, analytical chemistry, and immunochemistry, to mention the most relevant ones. Clinical chemistry provides the fundamental support to physicians and healthcare providers to improve disease diagnosis, exclude the pathological conditions, and to formulate therapeutic treatments. The widespread technologies in clinical chemistry Liquid Chromatography. http://dx.doi.org/10.1016/B978-0-12-805392-8.00022-0 © 2017 Elsevier Inc. All rights reserved.

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include colorimetry, spectrophotometry, potentiometry, fluorimetry, chemiluminescence, and so on, in most cases applied to biochemical, enzymic, and ligand assay methodologies, i.e., immunoassays. The limitations of immunoassays which oppose the detection of low-molecular weight compounds narrows down their fields of application. Moreover, flexibility is rather restricted as analyses of interest rely on the availability of commercial or in-house validated immunoassays. The major challenge faced by the clinical laboratory is automation to cut the turnaround time, accelerate diagnosis, and enhance preventive medicine. The technical development achieved in mass spectrometry (MS) over the last decades has opened novel perspectives in several clinical fields. MS applications were pioneered in biochemical genetics and in the diagnosis of inborn errors of metabolism [1]. MS technology was then extended to clinical and forensic toxicology [2]. The successful coupling of MS with liquid chromatography (LC-MS) achieved by means of electrospray ion (ESI) sources has generated a great impulse in the development of LC-MS-based assays to be transferred to clinical chemistry. In a virtuous progression, LC with tandem MS (LC-MS/ MS) is now commonly used in clinical specialties such as metabolic disorders, endocrinology, and therapeutic drug monitoring. In the clinical laboratory, LC-MS finds its ideal space in the analysis of particularly challenging metabolites, such as steroids, amino acids, eicosanoids, and so on. Excluding the exogenous compounds, clinical chemistry faces an enormous concentration range of endogenous metabolites in biofluids (Fig. 22.1) [3]. LC-MS, more than other technologies, meets the criteria of wide dynamic concentration ranges and flexibility required in clinical chemistry. Nowadays, the strength and the weakness of the LC-MS technology have become apparent together with its challenges and future opportunities. The reader can find a large body of literature that revises the present and the future of LC-MS in clinical chemistry. This section covers several aspects of the LC-MS-based assays, organized in the manner of standard workflows from sample manipulation, to separation strategies, to detection methodologies, that can be generalized to many areas of clinical LC-MS applications.

22.2 ­SAMPLE PREPARATION IN CLINICAL LC-MS LC-MS assays can be susceptible to interferences from high matrix content. This is involved with pitfalls that may affect the results. Ion suppression is the major issue that impacts mainly selectivity and sensitivity, particularly when high-resolution-MS (HR-MS) engines are used. Polar lipids such as phospholipids in the biofluids or chemicals such as anticoagulants or plasticizers from test tubes can deposit on the stationary phase of the LC column and in the MS/MS interface region causing either suppression or enhancement of ionization [4,5]. Sample pretreatment improves sensitivity and removes interferences from the matrix preventing deleterious effects in the LC-MS system and eventually leading to improved data quality. The choice of the extraction method depends on several factors, including the chemical properties of the analytes, costs

22.2 ­ Sample preparation in clinical LC-MS

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Cholesterol Phosphatidylcholine Triglycerides Glucose Acetylcarnitine

Dynamic concentration range

Free fatty acids

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Lactate Fructose Octanoylcarnitine Galactose Uric acid Alanine Bilirubin Arginine Citric acid Phenylalanine Leucine Creatinine Isoleucine Sphingolipids Pyruvate Phytosterols Arachidonic acid Hydroxyproline Homocysteine

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Urea

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Methylmalonic acid 25-Hydroxycalciferol Endocannabinoids Dehydroepiandrostenedione

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Metanephrine Adrenaline 1,25-Dihydroxycholecalciferol Estradiol Prostaglandin E2 , Isoprostanes , Leukotriene B4

Plasma/serum metabolites

FIG. 22.1 The range of concentrations of endogenous metabolites in serum or plasma determined by targeted or untargeted LC-MS assays. From Becker S, Kortz L, Helmschrodt C, Thiery J, Ceglarek U. LC-MS-based metabolomics in the clinical laboratory. J Chromatogr B Analyt Technol Biomed Life Sci 2012;883–884:68–75.

of consumables, and involved time. Many procedures that can be done separately or in a combined manner are available to perform sample purification. The most widely used ones are protein precipitation (PP), liquid-liquid extraction (LLE), and online or offline solid-phase extraction (SPE) that can be preceded by clean-up steps such as filtration or centrifugation. Internal standard (ISTD) addition is recommended to control the recovery or loss of the initial amounts of the analytes during the extraction process. The IS added in advance in sample preparation can be exploited to normalize the response and correct the effect of ion suppression when analytes are then detected. Indeed, the compounds of the biological matrix and the ISTD undergo the same process, from sample extraction through MS acquisition. The analyte-to-ISTD response ratio compensates for the occurrence of ion suppression, providing a more accurate and precise data acquisition. PP is the fastest extraction method for an appreciable removal of proteins that are troublesome in many respects. In the majority of cases, PP is achieved with refrigerated methanol or acetonitrile. Simultaneously, soluble molecules are dissolved in the solvent used for the PP. The disadvantages of PP consist of poor recoveries of target compounds, partial protein removal, and ion suppression [6,7]. LLE, also known as solvent extraction, is based on the use of two different immiscible liquids, usually water and pure or mixed organic solvents. LLE exploits the

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partitioning properties of molecules between the two phases [8]. LLE is the preferred method in targeted analyses in urine, blood (serum or plasma), and other body fluids. The recovered organic phase, which can be the bottom or the upper layer, enriched in the analytes is dried under inert gas, typically nitrogen. The advantages of the LLE are the requirement of simple laboratory equipment, whereas the disadvantages are represented by large solvent consumption and time required for evaporation. By means of liquid handlers both LLE and PP can be partially or fully automated. The widely used SPE provides clean extracts, protein removal, compound concentration, and high recoveries. In some applications, SPE is preceded by either PP or LLE [7,9]. SPE can be partly or fully automated by using high-throughput workstations or simpler and less expensive vacuum manifolds. The analytes are separated from the matrix through the passage onto a stationary-phase packed in a cartridge. The choice of optimal cartridge geometry, depending on the available volume, the sample number, and the budget, is critical for the productivity of the laboratory. The most commonly used cartridges are shaped as packed syringes or 96-well plates. Miniaturized cartridges are suitable when limited amounts of the biological fluids are available for online sample preparation. Microcartridges have been designed to achieve extraction on miniaturized devices such as microextraction by packed sorbent (MEPS) [10,11], solid-phase microextraction (SPME), and liquid-phase microextraction (LPME) [12,13]. Several types of stationary phases can be apt to the specific analytical requirements: normal phase (NP), reversed phase (RP), and ion exchange (IE). Normally, SPE extraction comprises a series of steps, in the sequence: cartridge equilibration, washes, sample load, sample matrix washout, and eventually compound elution. SPE online or column switching is performed directly on the LC system where an SPE column, shaped as a cartridge or a disk, is placed before the separation column. This allows for the entire or partial SPE eluate to be selectively transferred to the analytical column by switching valves [14–16]. Fig. 22.2 provides a schematic representation of the operational steps of the SPE online.

22.3 ­LC-MS TECHNOLOGIES IN CLINICAL CHEMISTRY 22.3.1 ­LIQUID CHROMATOGRAPHY LC is a potentially universal technique for the separation of compounds from mixtures. Since its introduction in the clinical chemistry, the availability of stationary phases has expanded the application possibilities to biomolecules with very different characteristics. Column manufacturing technologies range from the packing with regularly or irregularly shaped particles to monolithic porous material. The former ones are the conventional stationary phases where the solid material is discrete as it consists of spheres with or without a solid core. Monolithic columns are a single piece of porous cross-linked polymer or porous silica with a bimodal structure consisting of macropores and mesopores. The larger macropores (around 2 μm) ­allow the solvent to pass through the column generating less flow resistance, whereas

22.3 ­ LC-MS technologies in clinical chemistry

Position 1–Loading/cleanup

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FIG. 22.2 Schematic representation of an on-line SPE for the LC-MS system. In the load/clean-up position, pump A loads the sample onto the SPE cartridge, while pump B delivers the eluents to the LC-MS system. In the SPE clean-up, analytes are retained by the SPE stationary phase, while unwanted constituents are delivered to the waste. In the separation/elution pump B assists the elution of analytes off the SPE and the eluates are introduced into the LC-MS apparatus.

mesopores (around 13 nm) are smaller and allow the analyte separation to occur. As against a packed column, the manufacture of monolithic phases attempts to optimize mass transfer kinetics [17]. Furthermore, the column materials provide better stability, shorter retention times (RT), longer column lifetime, and satisfactory resolution compared with conventional columns packed with particles [18]. Importantly, monolithic columns are compatible with conventional HPLC [19]. A possible disadvantage is the consumption of large volumes of mobile-phase. HPLC performances influence the choice of column geometry and technologies. For example, the UHPLC system operates up to 20,000 psi [1400 bar] of backpressure making it possible to use columns packed with sub-2-μm porous particles. Instead, the conventional HPLC systems operate at about 6000 psi [400 bar] and require column with more porous particles than the UHPLC column. Currently, the majority of small molecules in clinical chemistry are analyzed using bonded-phase packing in particular: NP and RP.

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In NP chromatography the stationary phase is polar and the mobile phase is nonpolar or moderately polar. The rate at which individual solutes migrate through NP columns is primarily a function of their polarity. A typical stationary phase for NP chromatography is silica. There are also bonded NP materials. They have organic moieties with cyano and amino functional groups. Mobile phases most frequently used in NP consist of hexane, isooctane, or chloroform as the prevalent part, which can be added to trifluoracetic acid or acetic acid in the range 0.1%–0.4%. Isocratic elution is the preferential choice in NP separations as the gradient elution is affected by poor reproducibility due to unpredictable mechanisms of absorption of polar solvents and traces of water to the stationary phase, which cause fluctuations in the analyte partition [20]. Hydrophilic interaction chromatography (HILIC) is a variant of NP chromatography in which the retention mechanisms are simultaneous IE, partitioning, and hydrogen binding resulting in the enhanced RT for polar analytes. The mobile phase used in HILIC is highly organic in nature, typically acetonitrile containing a small percentage of aqueous or other polar solvents. HILIC stationary phase is polar and the aqueous portion of the mobile phase acts as the stronger solvent, which is opposite to the conventional RP chromatography [21]. There are many types of HILIC stationary phases: bare/hybrid and amide-bonded silica, then diol, zwitterionic, aminopropyl, and the cyano ones. Emphasis of advantages is given when HILIC is coupled with ESI/MS detection by virtue of the more efficient desolvation in the ion source of the highly organic mobile phases used. Furthermore, low backpressures are observed in the HILIC system, due to the low viscosity of the organic-rich mobile phases. The main constraints of HILIC are due to lack of retention of acidic as well as neutral compounds. Moreover, limitations consist of longer equilibration times of the column and distortion of peak shape that arises from the mismatch of sample solvent and mobile phases. Opposite to NP, RP separations involve nonpolar stationary phases and polar mobile phases; the latter ones typically composed of an aqueous eluent A and a monocomponent or multicomponent organic eluent B. The principle of separation is the partition of analytes between a polar mobile phase and a nonpolar (hydrophobic) stationary phase according to the compound-specific partition coefficient. The most popular nonpolar stationary phases result from binding octadecyl or octyl chains to the silica support to obtain C18 and C8 RP columns, respectively. An example of the mobile phase used in RP consists of water containing 0.1% formic acid or 5 mM ammonium formate as eluent A, and acetonitrile or methanol as eluent B, with or without the addition of 0.1% formic acid or 5 mM ammonium formate, consistently with eluent A. Often, a small part of the organic solvent is added to the mobile-phase A. Gradient elution is widely used for the separation of complex mixtures. A minimal part of the aqueous phase (at least 1%) should be kept at the maximum of the gradient to grant ionization even when the totality of the organic phase is reached. The solvent type influences the response of compounds in the ESI source. For example, methanol and acetonitrile play a relevant role in the ESI ionization processes of hormones [22]. The elution order of the solutes in a mixture is governed by their hydrophobicity. RP is advantageous for separating mixtures of homologous series in which components differ in molecular

22.3 ­ LC-MS technologies in clinical chemistry

weight and/or water solubility. RP is the elective chromatographic technique in clinical applications. Pentafluorophenyl columns (PFP) deserve a special mention. The derivatization of silica with pentafluorophenyl headgroups masks hydrogen donor groups and limits hydrogen binding providing a high degree of steric selectivity to separate structural isomers. The electronegative fluorine groups offer particularly high selectivity for cationic compounds. PFP stationary phases are widely used for the analysis of hormones (see below). Regardless of the chemistry of the stationary phase, it is a good practice to use a guard column before the analytical column to extend its lifetime. Normally, guard columns have the same or similar stationary phase as the analytical column. Their role is to trap particulate and those compounds that have a strong interaction with the stationary phase and that after many runs can increase the backpressure, background, and compound resolution.

22.3.2 ­ LC-MS INTERFACES Coupling the LC system with MS is accountable among the most revolutionary changes introduced in analytical chemistry. To be measured by MS, eluted analytes, mostly low-molecular weight molecules, need to be ionized and transferred from the dissolved status into the gaseous phase. The ESI source and the atmospheric pressure chemical ionization (APCI) source accomplish simultaneous ionization and desolvation of molecular ions and represent the most common interfaces between HPLC and MS. ESI and APCI are soft ionization techniques associated with improved analyte detection. Minimization of inaccuracy and gross errors associated with ESI and APCI facilitates identification and expansion of the application fields of LC-MS detection. The charged molecular ion species are generated by different ionization mechanisms in ESI and APCI [23,24]. In these two ionization techniques, the temperature of the source plays an important role in solvent evaporation and mass ionization process. The choice of source temperature is a function of the solvent type and thermostability of the metabolites. The degree of ionization influences the sensitivity of MS detection. The majority of LC-MS applications are performed with the ESI source. ESI has the advantage of limiting the occurrence of artifacts. Moreover, ESI is associated with low fragmentation rates and may lead to multiple charged ions. Protonated and deprotonated ion molecules, [M+H]+ and [M−H]−, respectively, are produced more efficiently in the ESI source. However, [M+Na]+, [M+K]+, [M+NH4]+, [M+Li]+, [M+Cl]−, [M+HCOO]−, and [M+CH3COO]− ions are frequently observed depending on the salts and modifiers added to the mobile phase. Additional ions result from the noncovalent interactions between the produced ions and the neutral solvent molecules. In many cases the formation of adducts is the only way of detecting molecules with a scarce tendency to acquire or loose protons. Noteworthy, multiple adducts formed by the same compound contribute to sample identification. Initial solvent composition and eluent changes during the chromatographic gradient influence the formation of cluster ions. In particular, high water percentage causes poor evaporation and ionization and spray instability. In contrast, as mentioned earlier, a small percentage of the aqueous eluent should be maintained in the late gradient to allow

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ionization of last eluted compounds. The ionization process of the APCI is more energetic than ESI as the ion transfer occurs through corona needle discharges that generally favor singly charged ions. The APCI is less susceptible to ion suppression than ESI because analytes are efficiently transferred into the gas phase when ionization occurs. Furthermore, APCI favors the ionization of low-to-medium polarity compounds bearing atoms with high proton affinity. In contrast, APCI suffers from lower sensitivity and has limited applications to the analysis of thermally unstable compounds. Atmospheric pressure photoionization (APPI) has been developed more recently than ESI and APCI. APPI suffers from reduced matrix effects. In the APPI source, the LC eluent is nebulized into a heated probe where a “dopant” compound is evaporated and ionized by ultraviolet radiation (UV) to form photo-ions. Several factors have likely limited the acceptance of APPI, including commercial ones and competing spaces with the more widespread ion sources. The recent developments might in future increase acceptance and spread applications of APPI [25,26]. On the basis of the operational principles of ESI and APCI several variants with different geometry and properties of these ion sources have been designed to improve performance. Nanospray has resulted from the miniaturization of ESI to reduce flow rates and the droplet size for a more efficient ionization. Coupling chip-based nano-LC to MS with a nanospray allows detection of low-abundance metabolites, e.g., 8-isoPGF(2α) in human urine for diagnostic purposes [27]. Recently, engineering of the ESI interface with droplet-based microfluidics has proven promising in the measurement of enzymic kinetics and screening for inhibitors [28].

22.3.3 ­MASS SPECTROMETERS MS measures the mass-to-charge (m/z) ratio of molecules that have been converted from their charged state into the ion source. When in-source fragmentation occurs, the MS detector measures molecular and fragment ions produced during the ionization process. MS technology enables the development of flexible and robust methods and the simultaneous quantitation of low- and high-molecular weight analytes, even in a wide range of concentrations. Thus, in contrast to other detection systems, MS allows for the analysis of multianalyte panels using a single method, saving time and resources. Several technologies are available for the detection of ions, which combined with the ion source technologies to expand the number of possible combinations between ion sources and MS detectors, mainly quadrupole, time of flight (TOF), and hybrid analyzers. When reduced to practice, only a few configurations respond to the versatility required in clinical chemistry. At the present time, LC coupled to tandem MS (LC-MS/MS) using triple quadrupole MS (QqQ) finds widespread application in the field of clinical biochemistry. In the last decade, the advances in MS technologies and in particular in HR-MS offer new opportunities for the detection of analytes combining sensitivity and wide dynamic ranges of QqQ instruments with high resolution and mass accuracy. The downsides are their high complexity and management of the technologically advanced equipment.

22.3 ­ LC-MS technologies in clinical chemistry

22.3.4 ­ QUADRUPOLE ANALYZERS Quadrupole analyzers are low-resolution mass analyzers that usually operate at unitary mass resolution. The upper detection limit of a quadrupole is usually at 400 amu. Quadrupoles can be set to detect ions within the detection range (scan mode) or to monitor a specific m/z value, a setting known as single ion monitoring (SIM) that lowers the detection limits of target analytes. Controlled fragmentation is not achievable with a single quadrupole analyzer. However, ion fragmentation can be induced by applying a high voltage between the source and the skimmer lenses before the detector in a process known as collision-induced-dissociation (CID) [29]. Parameters of this process can be controlled when CID occurs in a collision cell, which is a quadrupole designed to maintain the collision gas at a low pressure and to transmit the fragment ions produced. The principle of tandem MS is based on the use of configurations of two mass analyzers arranged in series which interposed a collision cell. The combination of two quadrupole mass analyzers with the collision cell is named QqQ MS. In tandem MS two or more stages of MS analysis are independently applied, providing the advantage of increased specificity over single-stage MS. The mass filters are used in static or scanning mode to select a single m/z or to acquire an m/z range. Ions of a particular m/z (precursor ion) can be selected in the first mass filter. The second mass filter is set to select the fragment ions (product ion) that are formed from fragmentation of these ions occurred in the collision cell after collision with inert gas molecules. This MS experiment provides a spectral fingerprint of molecules and constitutes the basis of single reaction monitoring (SRM). In this experiment, the first mass filter is set to a constant m/z, the second filter can be set to the m/z of a particular fragment ion. The major MS experiment used in clinical chemistry is called multiple reaction monitoring (MRM) in which many transitions (reaction pairs) are monitored. MRM experiments reduce the background signal, increase the duty cycle (the measure of the instrument's time actually devoted to measuring signals) of the instrument, resulting in sensitivity gain and dynamic range extension.

22.3.5 ­ ACCURATE MASS ANALYZERS Various accurate mass analyzers are available ranging from single-stage HR-MS (i.e., TOF and Orbitrap) to powerful hybrid analyzers that couple quadruple technologies with HR-MS. Hybrid HR-MS analyzers (e.g., QqTOF/MS) comprise a quadrupole, a collision cell, and an HR-MS analyzer in sequence to achieve high sensitivity, mass resolution, and mass accuracy for the parent and fragment ions. In addition, HR-MS/MS produces the full product ion spectrum, enhancing the information content. These advantages are partially reduced by the lower sensitivity of the MS/MS transition [30]. When HR-MS is combined with UHPLC, the potential for an enhanced acquisition speed provides a high-end tool for clinical diagnosis. HR-MS engines have the advantage of detecting the full mass spectrum simultaneously with excellent mass accuracy (<2 ppm), providing a better overview of the entire sample extract. Analytes identification is performed by matching the measured accurate mass and isotopic pattern with the elemental composition of the candidate

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compounds. In the past, low-resolution instruments imposed several limitations compared with HR-MS instruments. The recently developed single-stage HR-MS engines provide mass resolutions ranging from 20,000 up-to 500,000 FWHM. Today, numerous reports show that the quantitative performance of most HR-MS is similar to QqQ [31,32]. The real advantage to adopt HR-MS technologies is provided by the improved isomer resolution and the extension of the detection coverage. This can assist the clinical need for a comprehensive screening of endogenous or exogenous metabolites allowing retrospective data treatment crucial in legal issues. In contrast, the high initial cost of instrument is a critical implication.

22.4 ­APPLICATIONS 22.4.1 ­FAT-SOLUBLE VITAMINS General Considerations—Vitamin A (beta-carotene, retinol, retinal, and retinoic acid), vitamin D (cholecalciferol and ergocalciferol), and vitamin E (tocopherols and tocotrienols) are groups of fat-soluble low-molecular weight compounds playing multiple biological roles in the human body [33]. Conversely, vitamin K, which includes phylloquinone and menaquinone, has a narrower spectrum of biological effects, acting as a cofactor of enzymes that catalyze the carboxylation of the glutamate residues of proteins involved in the coagulation cascade and the synthesis of osteocalcin [34]. Since both the blood coagulation testing and quantitation of the circulating osteocalcin are clinically more useful than the direct vitamin K measurement, this paragraph will touch exclusively upon the analysis of A, D, and E vitamins forming the so-called ADE complex. From a clinical standpoint, the assessment of vitamin D represents the most frequently prescribed one among fat-soluble vitamins in the general population, while vitamins A and E are usually investigated within a specific diagnostic issue [35]. Therefore, the scientific literature enlists tens of LC-MS/MS methods developed for measuring vitamin D metabolites, while to date it is possible to quote only a couple of papers describing a validated method for vitamins A and E assessment in serum [36–38]. Notably, due to their common lipophilic nature, a single chromatographic method can be developed to assay the whole ADE complex within the same batch [39]. It should be noticed that while the routine measurement of major vitamin D metabolites can take just a few minutes, a comprehensive method for the ADE complex can run up to 30 min. Moreover, comprehensive methods necessitate adequate instrumentations, in terms of chromatographic precision and MS sensitivity, since the range of concentrations to be covered spans from picograms of calcitriol up to micrograms of retinol and tocopherol [33]. Thus, the choice of the appropriate analytical setup must be tailored to the actual laboratory throughput rather than to the potentialities of a comprehensive method. Preanalytics—The analytes of the ADE complex show good stability in whole blood, serum, and plasma for up to 1 week if stored at room temperature or +4°C, regardless of the light exposure [40]. In this regard, one action to take is to protect the samples after matrix extraction, when the analytes have been displaced from their

22.4 ­ Applications

shielding carrier complex. It should be remarked that stability means that the loss of analytes due to degradation biases the assay less than the joint effect of biological variability and analytical imprecision. Serum and plasma are comparably suitable for ADE complex analysis [41,42]. However, in the range of vitamin D metabolites, plasma samples show significantly higher calcitriol level compared with serum, suggesting this matrix as suitable for accurate measurement of active vitamin D load [42]. Sample Preparation—Freeing fat-soluble molecules from their carrier protein is a pivotal step in their accurate quantitative analysis. LLE is a suitable strategy for the extraction of the ADE complex from biofluids [39,43]. Basically, a volume of sample (as small as 0,1 mL depending on LC-MS/MS apparatus performance) is diluted 1:1.5–2 volumes with methanol to achieve PP; after centrifugation the liquid phase is transferred and mixed with an excess volume (3–6 volumes) of n-hexane, diethyl ether, or ethyl acetate. The organic phase is recovered and dried under nitrogen stream, and the extract is reconstituted into the appropriate loading phase (usually methanol, between 0.1 and 1 volume of the initial sample volume depending on the apparatus performance). All the passages have to be carried out in glass tubes and amber vials under dim light. Alternatively, for large batches of samples, the LLE can be replaced with a more productive SLE using a multiwell plate and a robotic liquid handler to increase the throughput [38,44–46]. Offline SPE with C18 or C8 cartridges with a chromatographic bed of 500 to 10 mg can be alternatively used. While C18 offers a good general lipophilic profile which is suitable for fat-soluble vitamins; the hydroxyls residues in calcifediol and calcitriol make the C8 a better choice that allows to increase overall method sensitivity especially for calcitriol analysis [47]. Notably, sample preconcentration without affecting recovery is achieved with SPE, [43]. Preconcentration is particularly relevant if the method aims at measuring calcitriol, the values of which falls in the picogram range. Thus, if the SPE step is inserted online before the chromatographic analysis, the reached sensitivity is adequate to detect the low concentration of vitamin D metabolites [42]. Another major advantage of SPE on-line is provided by the elimination of most part of phospholipids, which are known to produce the matrix effect. Phospholipids are usually carried over by the LLE and partly by the offline SPE. Sample pretreatment is advantageous to improve the chromatographic performance and peak shape [39,48]. Remarkable gain in sensitivity has been observed by using immune extraction instead of classical “chemical” procedures in calcitriol analysis [49,50]. As it can be easily understood, this kind of approach targets a single specific analyte using the antibody affinity to enhance the separation of the analyte, but it is quite time consuming and comparably more expensive. Analytical Aspects—Challenges of the ADE complex analysis specifically consist in avoiding interferences arising from the isobaric compounds that can partly or entirely overlap target analytes. Indeed, it has been shown that using tandem MS does not warrant absolute specificity, unless the chromatographic design is optimized to resolve those stereoisomers indistinguishable on the basis of the mass fragmentation pattern [51]. Separation of calcifediol (25OH-D3) and calcitriol (1,25OH-D3)

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from their respective epimers (namely 3-epi-25OH-D3 and 3-epi-1,25OH-D3) is an issue since their respective biological contribution is still not completely understood [52]. Actually, epimers show an increased ionization efficiency (up to 135%), thus tending to disproportionate contribution to the calcifediol (and probably calcitriol too) mass peak which causes its overestimation [53,54]. Some have questioned the actual importance of epimers in the general population due to their small abundance (2%–6% area of unresolved mass peak), and to date they seem to be a potential issue only in the pediatric area where their contribution is quantitatively more relevant and clinically meaningful [55,56]. Similar considerations can be done for tocopherols, in which alpha, gamma, and delta isoforms necessitate to be resolved in order to avoid overestimation [57,58]. However, in a comprehensive method this kind of issue seems to be not an analytical concern [39,43]. Thus, the development of a comprehensive method for major ADE complex analytes (retinol, alpha-tocopherol, calcifediol, and calcitriol) can be based on the RP chemistry of a C18 chromatographic column, considering a PFP-bound phase when the resolution of isomers is mandatory. The mobile-phase system is usually made up with an acidic water solution (A) of ammonium acetate or formate (0.1%–0.2%, v/v) eventually with a modifier (e.g., 0.1%, v/v formic acid), and a methanol solution (B) with the same ammonium salt final concentration. Elution is achieved by means of a gradient, starting from >60% A and ending with almost 100% B, at a flow rate that usually ranges between 0.2 and 0.35 mL/min for a 2.1 × 100 (or 150) mm column with 2 μm or slightly larger bed particles and thermostated. Noteworthy, the expected order of elution in such conditions is roughly calcitriol → calcifediol → retinol → delta-tocopherol → alpha-tocopherol, with retinol tending to have an RT close to that of calcifediol in a >20 min runtime [39,43]. QqQ with the ESI source operating in positive mode has been successfully used in the large majority of methods published to date. Notably, the ESI source makes detection of the ADE analytes without prior derivatization, in that fat-soluble vitamins tend to ionize quite well. Indeed, the majority of them show the [M+H]+ molecular ion, while calcifediol, calcitriol, and alpha-tocopherol form a protonated precursor with a neutral water loss [M+H−H20]+. A summary of the mass transitions is provided in the bibliography [43]. It is generally recommended to avoid MRM transitions due to water loss for the quantitative analysis due to the instability and irreproducibility. In contrast, the detection of [M+H−H20]+ of vitamin D metabolites does not pose reproducibility issues [59]. Regarding mass transitions in MRM mode, due to the plethora of metabolites (especially for vitamin D) which may populate chromatograms in a comprehensive analysis, it is advisable to use both a quantifying and a qualifying transition in order to increase specificity in the recognition of each mass peak especially for short chromatographic separations where the resolution is suboptimal [39,46]. More recently, the availability of affordable hybrid HR-MS has offered an analytical alternative to the classical low-resolution QqQ for fat-soluble vitamin analysis [60,61]. Sensitive and accurate recognition of multiple metabolites and considerable shortening of the analytical time (about 5 min runtime) are accounted among the major advantages of HR-MS. Application of HR-MS to the analysis of ergocalciferol metabolites has

22.4 ­ Applications

shown no interference on cholecalciferol due to using suitable deuterated derivatives [60,62]. Nevertheless, the routine analysis of vitamin D is devoid of the above issues since the number of metabolites of real clinical interest is relatively limited and usually well resolved in fast chromatography. Additionally, most of the times ergocalciferol is undetectable and presents a clinical interest only in patients receiving oral supplementation. Indeed, HR-MS does not help to overcome the discrimination of epimers, which are resolved exclusively in the chromatographic compartment. Therefore, low-resolution MS/MS based on QqQ meets to a satisfactory extent the clinical operational requirements.

22.4.2 ­ POLYUNSATURATED FATTY ACIDS (AND THEIR METABOLITES) General Considerations—Fatty acids (FA) constitute a wide class of compounds which cover multiple biologically relevant functions, from fuelling cell metabolism through beta-oxidation, to supplying precursors for the synthesis of complex lipids (phospholipids, lipopolysaccharides, di-, and tri-acylglycerols) and signaling molecules (eicosanoids: prostaglandin, thromboxanes, leukotrienes) [63]. A large body of evidence demonstrate the pivotal role FA play in bridging chronic inflammatory diseases with metabolic disorders, with type 2 diabetes mellitus being the most important candidate pathology model [64]. In particular, the essential polyunsaturated FA (PUFA) linoleic acid (LA) and alpha-linolenic acid (ALA) represent the precursors of proinflammatory omega-6 (n6) and antiinflammatory omega-3 (n3) eicosanoids, respectively [65]. Notably, the dietary intake of n3 has been shown to inhibit the endogenous n6 synthesis, thus allowing the control of inflammatory triggers via nonpharmacological interventions [66,67]. Thereby, for the growing interest shown in clinical chemistry, the analysis of nonerythrocyte PUFA will be the focus of the following section. Preanalytics—Two major issues are represented by autoxidation and hydrolysis. Free as well as bound PUFA are prone to peroxidation due to the presence of reactive methylene bridges (CH2) between double bonds (CC) in the carbon chain. Susceptibility to oxidation increases with increasing number of double bonds [68]. The formation of free radicals at the methylene position initiates a chain reaction involving the addition of molecular oxygen to form lipid peroxides or lipid peroxides (LPO). In turn, LPO as a form of reactive oxygen species (ROS), amplify and sustain the peroxidation process. On the other hand, hydrolysis is the process that causes FA release from membranes and alters the actual PUFA profile. In blood specimens, where about half of the volume is represented by erythrocytes, ferric hemoglobin (Hgb-Fe3+) represents a major trigger of oxygen radicals which causes PUFA peroxidation. Platelets, which are another abundant component of blood, represent a source of phospholipase activity that causes the hydrolysis of phospholipids. To prevent peroxidation and hydrolysis, plasma samples (collected preferably in EDTA) should be separated within 30 min from the cells and stored at −20°C for up to 12 days [69,70]. In contrast, samples stored at +4°C remain stable up to 48 h. Sample preservatives,

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which are useful for scavenging free iron (deferoxamine) and free radicals (butylated hydroxytoluene, BHT), deserve a special mention. Nevertheless, sample preservatives have proven poorly effective in the prevention of PUFA modifications. To date blood sampling tubes containing such preservatives are not commercially available [70]. Essentially, their use seems to be meaningful only for long-term storage of whole-blood samples, and not for clinical chemistry applications [69]. Sample Preparation—For nonerythrocyte PUFA it is meant that the amount of FA is not bound in the cell membrane. FA circulate unbound (nonesterified FA or NEFA) or bound as cholesteryl esters (CE), di- and tri-acylglycerols (DG, TG), and phospholipids (PL). Similar procedures of extraction are applied to biological specimens to address the analysis of FA in either their bound of free status. The aim is to extract free and bound FA from the protein matrix, mainly lipotroteins. Free FA (FFA) can be analyzed directly or after derivatization. In contrast, the analysis of bound FA requires a multiple step sample preparation where extraction is simultaneous to or followed by hydrolysis, which is fundamental to recover FA. Basically, the extraction procedure consists in de-esterifying FA using a heated organic diluted solution of alkali (usually 0.3 M KOH in ethanol or methanol), whose major advantage is to produce an irreversible reaction (also known as saponification) [71]. Once the reaction has taken place (always in Pyrex-glass vials) and the vial has been cooled down, an organic solvent (n-hexane) is added in slight excess, thoroughly mixed, and then separated by centrifugation. The organic layer is finally transferred, dried under a nitrogen stream, and reconstituted adding a methanol/water solution [72]. Notably, the method is suitable for 0.1–0.2 mL of sample (which should be already added with BHT to prevent peroxidation during the extractive process). The most popular method for the extraction of free and bound PUFA is the Bligh and Dyer's procedure [73]. Several adaptations to Bligh and Dyer's method can be found in the literature; however, it still has the disadvantage of using toxic solvents. Either offline or online SPE procedures based on mixed-mode reversed-phase/strong anion-exchange polymeric sorbent [74], and RP C18 sorbent, respectively, have been developed to separate fats from the biofluids [75,76]. Both allow a comprehensive analysis of PUFA alongside the cascade of their metabolites in that the SPE is more suitable for hydroxylated compounds than the organic solvent procedure previously described. SPE is recommended for investigating the inflammatory cascade rather than the total PUFA profile. Analytical Aspects—Chromatography can be achieved in RP mode on a C18 column, using either isocratic or gradient elution. An example of the chromatographic isocratic is given by the binary system of water/acetonitrile with ammonium salt (A) and water/2-propanol with the same final salt concentration (B) in a 3:1 ratio [77]. Gradient elution compatible with both extraction/saponification and SPE can be achieved with an ammonium salt solution (A) and acetonitrile (B) system, which are modified changing the composition of the phases with addition of the acetonitrile/2propanol mixture in order to combine with the on-line extraction system [72,74,75]. Diluted formic acid (0.2%, v/v) can be used as a modifier. On average, chromatographic separation is achieved within 10 min using a 100 × 2.1 mm column with 2 μm

22.4 ­ Applications

particle size at a 0.2–0.3 mL/min flow rate, and a column temperature thermostated at 40°C. In comprehensive methods that cover the metabolite cascade of PUFA, the elution order in a water/acetonitrile chromatographic system is usually prostaglandins/ thromboxanes → leukotrienes → hydroxy-PUFA → PUFA [74]. In GC-MS, which has been the reference analytical approach until recently, FA are analyzed after transesterification to form FA methyl esters (FAME) [78]. It is noteworthy that such a chemical process has some caveats, e.g., long-chain FA (>24 carbon atoms) are scarcely volatile even after their conversion to methyl esters. Conversely, LC-MS/ MS allows the direct analysis of underivatized FA using either ESI or APCI ion sources [79]. Owing to the carboxylic acid moiety, PUFA and their metabolites tend to form the [M−H]− and [M−H2O−H]− precursor ions in negative mode [75,77]. The detection of FFA in positive ion mode is driven by the addition of ammonium salts in the mobile phase [80]. It must be remarked that any comprehensive method has to cover ranges in limit of quantitation from micrograms for PUFA, to picograms for eicosanoid and other metabolites. The MRM mode achieved with a QqQ MS is an advantageous tool for the analysis of FA. The specific MRM transitions allow to discriminate the isobaric species that have a different disposition of double bounds in the side carbon chain due to the characteristic fragmentation pattern [81]. On average, the current methods using the MRM mode allow simultaneous assessment of up to 50 different analog compounds (PUFA and related metabolites) in a quite fast runtime (10 min) [74,75]. Notwithstanding, also in this field hybrid MS suited for quantitative HR-MS have been proposed for the analysis of PUFA and their metabolites [71]. Such a technology has been shown to be effective in metabolomics studies with in vivo administration of 13C-labeled compounds to track the routes of PUFA conversion. Overall, cost-effectiveness and robustness make QqQ MS preferable in the analysis of PUFA in clinical chemistry.

22.4.3 ­ ENDOGENOUS GLUCOCORTICOIDS General Aspects—Steroids represent a wide class of cholesterol-derived molecules which act as hormones controlling the sexual phenotype (testosterone, estrone, and estradiol), gestation (progesterone), electrolytes and water balance (aldosterone), anabolism and immunity (cortisone, cortisol and corticosterone). Among them, cortisone, cortisol, and corticosterone (collectively named glucocorticoids) are of fundamental clinical importance being involved in a number of endocrine disorders (namely hypocortisolism and hypercortisolism syndromes), with their analogs of synthesis representing mainstay medications of acute and chronic inflammatory diseases. Moreover, steroids analysis is particularly relevant in toxicology and sports science, as they can be illicitly used for doping the athletic performance [82]. Preanalytics—Steroids and endogenous glucocorticoids can be assayed in different biological matrices, namely blood, urine, and saliva as well [83]. Noteworthy, hair are suitable for nonroutine retrospective/forensic analysis [84–86]. In general, steroids show a considerable stability in whole blood, serum, and plasma and do not

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seem to necessitate a prompt separation after sample collection [87,88]. Thereby, serum specimens can be considered stable up to 72 h at RT, and up to 1 month at −20°C, with a good resistance to multiple freeze-thaw cycles [87]. The same characteristics of stability can be stated also for saliva samples, with no significant differences between frozen and RT samples [89]. It should be reminded that up to 95% of endogenous glucocorticoids are bound to high-affinity binding proteins, so that just the unbound 5% fraction undergoes glomerular filtration and resorption [90]. Therefore, both spot and 24-h urine samples are suitable for free corticosteroids analysis, but only the latter is clinically meaningful [91]. Noteworthy, similar to what is seen above, urinary glucocorticoids are stable for more than 72 h regardless to the preservative used and the storage temperature (room temperature, chilled, or below zero) [92,93]. Sample Preparation—For glucocorticoid analysis in serum or plasma a simple extraction based on PP with zinc sulfate or organic solvents such as acetonitrile is used, considering that the endogenous level allows to reach an acceptable signalto-noise ratio [94]. However, in order to develop a comprehensive method, PP must be refined using an LLE with stronger organic solvents (e.g., methylene chloride), or replaced by SPE to achieve a dimmer chemical background while improving the recovery of more hydrophilic compounds [94]. Clearly, a general purpose method for sample purification cannot be achieved, and rather the sample clean-up must be developed considering the specific necessities as well as analytical objectives. Although urine and saliva are matrices of relatively low chemical complexity to assess free steroids, they oppose a certain interfering background and usually contain analytes at a considerably low concentration [94]. Furthermore, in saliva, mucins may require a physical or chemical disruption in order to preserve chromatographic apparatus integrity [95]. Similarly, the enzymatic digestion of urines may be necessary to free steroid metabolites from their glucuronic conjugation, in order to allow a comprehensive metabolites analysis [96]. Notwithstanding, as shown for saliva analysis, it is possible to achieve both sensitivity by means of SPE and automation using a robotic liquid handler that performs an offline procedure using 96-well microelution plates [97]. Furthermore, SLE is suitable for steroid extraction as it gives cleaner eluates and reduces the emulsion of samples. However, its lower recovery efficiency restricts its use to the analysis of more abundant molecules [94]. Analytical Aspects—As seen for other applications in previous sections, a single general purpose method for steroids analysis cannot be described. The literature is rich in papers reporting different chromatographic conditions in terms of chemistry of columns and mobile phases [94,95]. Notwithstanding, the majority of methods developed for endogenous glucocorticoid analysis in serum, urine, and saliva relies on C18 columns and methanol with ammonium salt and/or formic acid running in gradient mode [98]. Obviously, the major concern is that the chromatographic method must ensure robustness and reliability, especially with respect to the resolution of isobaric and isomeric compounds that may flaw specificity of mass analysis. Such an issue must be further considered with respect to the sample matrix used. For instance, in serum or plasma a major challenge is the separation of corticosterone from 11- and

22.5 ­ Future perspectives

21-deoxycortisol, which differs in the position of a single hydroxyl moiety on the four-ring core [94]. Conversely, in urine a challenge is opposed by the separation of tetra-hydrocortisol from allo-tetra-hydrocortisol, which are excreted metabolites of cortisol [96]. If an UPLC apparatus is unavailable to pursue physical separation using sub-2-μm size bed particles, it is possible to use alternative column chemistries, such as the phenyl or bi-phenyl, which deploy the pi-pi interaction, or bed structure like fused-core particle columns [99,100]. The ESI source operated in positive mode is widely used for steroids and glucocorticoid analysis, so mass analysis tends to rely on the [M+H]+ precursor ion when a protic organic solvent (methanol) as well as the proton supplier (ammonium salt, formate or acetate) is in the mobile phase [98]. The APCI is also a valuable alternative to ESI, and has been shown to be particularly effective in ionizing such ionization-refractory molecules such as cortisone and producing an attenuated chemical background [95,101]. Generally speaking, this is not critical for glucocorticoids, since differences in the background noise probably depend on the manufacture of ion sources rather than the ionization process itself. Although the QqQ MS is considered a specific mass analyzer, there are some specific issues to take into consideration when setting up a quantitative method for steroids and particularly glucocorticoids [94,98,99,102]. For instance, the choice of the most intense and thus sensitive MRM transition may not correspond to a similarly adequate specific recognition, as observed for cortisol when prednisolone and its metabolites were simultaneously present [98]. Moreover, more than one mass transition could be necessary to monitor endogenous glucocorticoids due to the presence of exogenous ones, and vice versa [102]. Finally, the choice of the appropriate surrogate ISTD labeled with deuterium is critical to avoid overlaps in the mass fragmentation pattern due to the naturally occurring isotopes. As a concluding and general note, steroid analysis can benefit from chemical derivatization to enhance the ESI process [103]. Particularly, such a procedure has been shown suitable for the comprehensive corticosteroid analysis in saliva [104].

22.5 ­FUTURE PERSPECTIVES The strengths of LC-MS and LC-MS/MS include the wide range of analytes that can be detected and measured simultaneously allowing multiplexed analyses. Although the initial costs of equipment are high, widening the coverage of comprehensive assessments in clinical chemistry means faster capitalization of investment. Importantly, the risk assessment of cardiovascular disorders, which are among the leading causes of death, would benefit from accurate definition of the dyslipidemic status. A multiplexed LC-MS/MS assay has been validated and automated to assist phenotyping and quantitation of apolipoproteins [105]. The development of methods for metabolic profiling is becoming an intense research activity due to the enormous implications in several areas of the diagnostic and personalized medicine. However, the release of clinically approved assays of metabolite profiles is limited by the regulatory constraints. To prepare our clinical mindset to extended metabolite profiles, it

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is recommended to read the recent review on the potential of steroidomics achieved by a metabolomics-based approach to measure comprehensively steroid perturbations in human beings [106]. Finally, even microbiology represents a new explorative area of LC-MS clinical applications [107].

22.6 ­CONCLUSIONS Improvements in the instrument configurations have enabled implementation of LC-MS into the routine clinical practice. Modern MS are highly sensitive and LC-MS methods are prospectively viable replacements for many immunoassays. Novel analytical strategies based on LC-MS and LC-MS/MS are emerging for the sensitive, quantitative multiresidual assays for metabolites simultaneously to their products of biotransformation. Multiple determinations of peptides in biofluids are a promising translation of the LC-MS universality into clinical applications [108,109]. The adaptations that have facilitated transferring LC-MS to the clinical laboratory reckon automated sample handling extended to sample extraction and derivatization, when applicable, improved sensitivity, instrument robustness, and reliability. The development of novel ionization methods and sources with elevated performances with the advantages of increasing the throughput and decreasing the maintenance will further extend the use of LC-MS from specialized operators to general medical technologists. Clinical biochemistry laboratories can largely advantage LC-MS assays when used for multiple and simultaneous detections with the potential for simplifying the laboratory setup and of enlarging test panels with a wide coverage of metabolite profiles.

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