Simultaneous determination of 12 vitamin D compounds in human serum using online sample preparation and liquid chromatography-tandem mass spectrometry

Accepted Manuscript Title: Simultaneous determination of 12 vitamin D compounds in human serum using online sample preparation and liquid chromatography – mass spectrometry Authors: Nur Sofiah Abu Kassim, Paul Nicholas Shaw, Amitha K. Hewavitharana PII: DOI: Reference:

S0021-9673(17)31772-7 https://doi.org/10.1016/j.chroma.2017.12.012 CHROMA 359068

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

24-8-2017 3-12-2017 5-12-2017

Please cite this article as: Kassim NSA, Shaw PN, Hewavitharana AK, Simultaneous determination of 12 vitamin D compounds in human serum using online sample preparation and liquid chromatography – mass spectrometry, Journal of Chromatography A (2010), https://doi.org/10.1016/j.chroma.2017.12.012 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.

Simultaneous determination of 12 vitamin D compounds in human serum using online sample preparation and liquid chromatography – mass spectrometry Nur Sofiah Abu Kassim, Paul Nicholas Shaw, and Amitha K. Hewavitharana* School of Pharmacy, The University of Queensland, QLD 4072, Australia *Corresponding author. Tel.: +61 7 334-61898; Fax: +61 7 334-61999

IP T

E-mail: [email protected]

U

Twelve vitamin D compounds (lipophilic and hydrophilic) quantified in a single run Vitamin D release from matrix through protein precipitation was optimised Inactive epimeric form of 25(OH)D3 was chromatographically separated Sensitivity improved by minimising matrix effects in MS using online sample cleanup Minimum sample preparation and on-line clean up: well suited for routine analysis

N

    

SC R

Highlights

A

Abstract

M

The development and validation of a method to simultaneously quantify 12 vitamin D compounds in human serum by LC-MS/MS is described. The main challenge was that of

TE D

extracting and chromatographing vitamin D compounds with a range of polarities, both lipophilic and hydrophilic, in a single analytical procedure. The extraction of all 12 vitamin D compounds were achieved by an optimised protein precipitation method using acetonitrile as

EP

the precipitant, and the separation was accomplished by using a pentafluorophenyl (PFP) column. The sensitivity was increased by minimising matrix effects in MS detector rather than using a lengthy derivatisation procedure; an online solid phase extraction (SPE) using a

CC

PFP guard column was used for cleanup. Detection limits for all compounds were in the picomole range when using a 500 µL sample volume. Recovery percentages ranged from

A

92% to 99%. LC-MS/MS resolution of all 12 vitamin D compounds, including the chromatographic separation of 25(OH)D3 from the isomer 3-epi-25(OH)D3 was achieved.

Stable isotope labelled vitamin D compounds were used as internal standards for the quantification of all 12 vitamin D compounds. This is a simple yet accurate, selective, and sensitive method for the quantification of 12 major vitamin D compounds, including the

1

sulfated forms, in human serum. The method is sufficiently robust to offer potential for use in routine analysis in a pathology laboratory setting. Keywords: vitamin D; vitamin D sulfate; LC-MS/MS; pentafluorophenyl (PFP) column; solid phase extraction (SPE)

Introduction

IP T

1.0

Recent literature has described contemporary mass spectrometry assays for the quantitative

SC R

measurement of multiple vitamin D compounds and their application in clinical research [1-

3]. It has been suggested that each member of the family of vitamin D compounds may have different functions beyond the traditionally referenced maintenance of musculoskeletal health towards conditions such as diabetes, cancer, multiple sclerosis, depression, and

U

cardiovascular disease [4, 5]. Consequently, the demand for measurements for vitamin D

N

compounds in pathology laboratories has significantly increased. Unfortunately, many studies

A

investigating links between vitamin D and diseases investigate a single metabolic oxidative pathway, that yielding 25-hydroxyvitamin D [25(OH)D3 and 25(OH)D2], to assess the

M

vitamin D status of individuals [6]. It is plausible that measuring only 25-hydroxyvitamin D concentrations will provide an incomplete picture of vitamin D status that may or may not

TE D

correlate with function or disease. An expansion of the range of vitamin D compounds that can be selectively quantified is hence important in the future for vitamin D research.

EP

Ideally, a benchmark method for all vitamin D metabolites would cover the relevant highand low abundance compounds, and would use universal, standardised analytical techniques

CC

[1]. Methods for the determination of lipophilic vitamin D compounds, especially 25(OH)D3, have been reported for a diverse range of biological fluids [7, 8]. However, the lack of analytical methods for the determination of the more hydrophilic vitamin D compounds, such

A

as the vitamin D-sulfate conjugate metabolites, has precluded their investigation in clinical

studies examining their role and biological functions. While controversy exists regarding their biological roles, it has been reported that 25-hydroxy D3-sulfate [25(OH)D3-S] may be a storage form of vitamin D3 and knowledge of its concentration may be expected to be helpful in the assessment of the vitamin D status, especially that of infants [9].

2

A previous method developed in our laboratory, using derivatisation methodologies, to determine major vitamin D compounds in human serum was found to be inappropriate for the determination of three of the sulfated (D2-sulfate, D3- sulfate and 25-hydroxy D2-sulfate) vitamin D compounds [10]. Later, a separate, non-derivatisation, method was developed to quantify sulfated forms alone, taking into account their hydrophilic nature [11]; both of these methods employed C18 columns. It was therefore resolved to undertake analytical method development to quantify simultaneously all 12 vitamin D compounds including the sulfated

IP T

forms [Vitamin D2 and D3 (D2 and D3), 25-hydroxy D2 and D3 (25(OH)D2 and 25(OH)D3), 24,25-dihydroxy D2 and D3 (24,25(OH)2D2 and 24,25(OH)2D3), 1,25-dihydroxy D2 and D3

SC R

(1,25(OH)2D2 and 1,25(OH)2D3), D2- sulfate and D3-sulfate (D2-S and D3-S) and 25-hydroxy D2-sulfate and D3-sulfate (25(OH)D2-S and 25(OH)D3-S)] using a simple method with no

derivatisation, incorporating some automation, and using a single column. The sensitivity was increased by reducing matrix effects in MS detector (using an additional sample clean up

U

step) instead of using pre-column derivatisation to improve ionisation in MS. The aim was to

N

develop a method that was sufficiently simple and robust to be used in the routine analysis in pathology laboratories to accurately quantify 12 vitamin D compounds, both lipophilic and

A

hydrophilic, using a single extraction and chromatographic run. The principal analytical

M

challenge was that of quantifying compounds with very different lipophilicities in a single

TE D

analysis procedure.

Vitamin D levels are often analysed in plasma or serum samples. A recent study, reporting on the determination of nine vitamin D compounds, revealed that serum levels of vitamin D compounds were higher than those in plasma samples taken at the same time [10]. Protein

EP

precipitation is a common approach used to release vitamin D compounds from serum or plasma binding proteins by using acetonitrile (ACN) or trichloroacetic acid (TCA) for protein

CC

denaturation [12]. However, establishing which protein precipitation method would release more vitamin D compounds and thereby yield a chromatographically cleaner extract has not

A

been studied to date. Protein precipitation does not remove phospholipids which can cause ion suppression in MS (thereby reducing sensitivity), can reduce HPLC column lifetime and increase the need for mass spectrometry maintenance due to contaminant build up on the ion source [13]. A sample clean-up technique such as solid phase extraction (SPE) is required to remove phospholipids from the extract before it is subjected to chromatographic separation. However, such a SPE clean-up procedure necessitates additional time and effort in method development. In the present study, two protein precipitation techniques were compared to be 3

used as the initial sample preparation method to remove biological proteinaceous matrices. An on-line SPE method using a guard column was also introduced to further clean-up the extract prior to LC-MS/MS separation. Stable isotope labelled analogues of each targeted compound (twelve in total) were used as internal standards to correct for matrix effects in the MS detector as well as to correct for the procedural errors during sample extraction and clean up.

IP T

To our knowledge, no LC–MS/MS method exists which is able to quantify simultaneously the lipophilic and hydrophilic vitamin D compounds in human serum; as stated above, currently available LC–MS/MS methods for the determination of sulfated vitamin D

SC R

compounds are limited only to the measurement of 25(OH)D3-S in human plasma (9) or separate quantification procedures are used for lipophilic and hydrophilic vitamin D

compounds, respectively [11, 14]. Hence, the aim of this study was to develop and validate a simple, effective and reliable method that can be used in a routine analytical laboratory to

U

quantify all major vitamin D compounds (both lipophilic and hydrophilic forms) in human

2.1.

Serum and plasma samples

M

Materials and methods

TE D

2.0.

A

N

serum, providing good precision, accuracy and sensitivity.

Blood samples were obtained from volunteer staff and research students (20 participants) of the School of Pharmacy, University of Queensland. Ethical approval was obtained from the

EP

Institutional Human Research Department, The University of Queensland. All volunteers provided written informed consent and relevant information such as the hours of exposure to

CC

sunlight during the week of interest, consumption of vitamin D supplements and the type. Serum samples were collected in vacutainer tubes without anticoagulant. Aliquots (500 µL)

A

of the serum samples were accurately measured and frozen at -80°C until analysis.

2.2.

Chemicals and reagents

All reagents and solvents were of analytical grade or LC/MS grade. Trichloroacetic acid (TCA) and 4-methylmorpholine were purchased from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Formic acid 4

(>99%) was purchased from Thermofisher (Geel, Belgium). High-purity water was prepared in-house using a Millipore Milli-Q system (Merck Millipore Billerica, MA, USA). Water and solvents for the mobile phases were filtered using a 0.2-μm Supelco Nylon 66 membrane from Supelco Inc (Bellefonte, PA, USA). Standard vitamin D compounds and several of the stable isotope labelled analogues [3-epi25(OH)D3, D2, D3, D2-S, D3-S, 25(OH)D2, 25(OH)D3, 25(OH)D2-S, 25(OH)D3-S,

IP T

24,25(OH)2D2, 24,25(OH)2D3, 1,25(OH)2D2, 1,25(OH)2D3, D2-d3, D3-d3, D2-S-d3, D3-S-d3, 25(OH)D2-S-d3, 25(OH)D3-S-d6, 24,25(OH)2D2-d3, 24,25(OH)2D3-d6, 3-epi-25(OH)D3-d3

SC R

and 1,25(OH)2D3-d3] were purchased from IsoScience (King of Prussia, PA, USA). Other labelled analogues (25(OH)D2-d6 and 25(OH)D3-d6) were purchased from Chemaphor (Ottawa, ON, Canada), (1,25(OH)2D2-d6) Medical Isotopes Inc. (Pelham, NH, USA), and (24,25(OH)2D2-d3) Toronto Research Chemicals Inc. (ON, Canada). Figure 1 shows the

U

chemical structures for all stable isotope labelled vitamin D compounds (for structures of the

A

Preparation of standard solutions

M

2.3.

N

analytes, the D’s should be respectively replaced by H’s).

All preparations and manipulations of vitamin D containing solutions were done under

TE D

subdued light. Stock solutions in ethanol of all standard compounds were prepared at a concentration of 1µM, were sub-divided into small volumes, and stored in amber Eppendorf tubes at -20 °C. All working solutions were prepared by serial dilution of stock solutions. The

2.4.

EP

combination standards contained the concentrations described in section 2.8 below. Preparation of sample and standard solutions prior to on-line SPE

CC

A frozen serum sample (500 µL) was thawed and spiked with 25 µL of the combined internal standard solution (to give the same concentrations as those in calibration standards in the

A

final solution injected onto LC-MS/MS – as listed in section 2.8), 975 µL of acetonitrile was added, and then vortex mixed for 1 min. The mixture was incubated for 15 min at room temperature to allow protein precipitation to be completed, then centrifuged at 4000g for 10 min. The supernatant (1100 μL) was transferred to a clean Eppendorf tube and mobile phase (75% B) added to a final volume of 1600 μL, and vortex mixed. The sample was injected using full loop mode with a 100 μL loop and overfill factor of 15. 5

With standards, 500 µL of each of the six combination standards containing internal standards was mixed with mobile phase (75% B) to a final volume of 1600 µL and vortex mixed for 1 min before injection.

Comparison of two solvents to release vitamin D from serum by protein precipitation

IP T

2.5.

The protein precipitation procedure used in this section was similar to our previous work [10]. Briefly, an aliquot of serum (250 µL) was mixed with 500 µL of acetonitrile or 2%

SC R

(w/v) trichloroacetic acid. The mixtures were vortex mixed for 30 s and then incubated for 15 min at room temperature. Following protein precipitation, the mixtures were centrifuged at 4000 x g for 10 min. The supernatants were transferred to clean Eppendorf tubes and

evaporated to dryness using a freeze drier (12 hr). The residues were reconstituted with 50 µL

U

of mobile phase (75% B) prior to injection of 20 µL in LC-MS analysis. In this experiment

N

the online sample clean-up was not used, and the extracts from two protein precipitation

A

methods were directly injected to the analytical column. Triplicate measurements were

2.6.

M

performed and peak areas were used to assess the efficacy of each set of conditions. Optimisation of on-line solid phase extraction (SPE) and separation

TE D

The optimisation of operating procedure was carried out using a combination standard prepared in section 2.8. Guard column (SPE) and analytical column were optimised separately, based on the highest peak areas and best peak resolution respectively. Parameters

EP

optimised were: stationary phase (PFP and C18), flow rate (0.2-1.2 mL/min), buffer (ammonium formate, 4-methylmorpholine) and mobile phase composition (25:75 – 60:40

CC

(water:methanol)). The on-line clean-up and separation procedure used in this study is depicted in figure 2.

A

2.7.

LC-MS/MS procedure

An Agilent binary LC system consisting of an Agilent 1290 infinity LC pump, Agilent 1290 well plate auto-sampler was used. A pentafluorophenyl (PFP) guard column (2.1 x 5 mm, 2.7 μm; Agilent Technologies, Santa Clara, CA, USA) and a PFP chromatographic column (150

6

x 2.1 mm, 2.7 μm (Agilent Technologies, Santa Clara, CA, USA) (column temperature 40 °C) were installed on the switching valve system as described in figure 2. For sample clean up and for chromatographic separation (pumps A and B) the same mobile phases were used: mobile phase A (aqueous 3 mM ammonium formate) and mobile phase B (methanol containing 3 mM ammonium formate). While operating in position A, the solid phase extraction (SPE) column (PFP guard column) is equilibrated with a mixture of the two

IP T

mobile phases in a ratio A:B (60:40), at a flow rate of 0.8 mL/min (using pump A) for 10 min. Then the sample (1500 µL), obtained following protein precipitation, was injected onto SPE and washed for 2.5 min with the same conditions.

SC R

During the period of SPE clean up, in position A, the analytical column was equilibrated

using pump B with the two mobile phases in a ratio A:B (75:25) at 0.2 mL/min. After 2.5 min wash, the guard column was connected to the analytical column using the switching valve

U

system to start the chromatography process (position B). The analytical mobile phase was

N

maintained for 11.5 mins at 75% B and the composition changed from 75 % B to 100 % B over the next 1 min, thereafter, it remained at 100% B for the next 9 min. The mobile phase

A

was then returned to the starting composition of 75 % B over the next 1 min, and the column

M

was subsequently re-equilibrated with 75 % B for 10 min before injecting the next sample. The total run time including the equilibration of the column was 35 min. During the

TE D

chromatographic run, the SPE guard column was washed using 100% mobile phase B using pump A.

An Agilent 6460 triple quadrupole tandem mass spectrometer equipped with a Jet Stream

EP

source and supported by Mass Hunter Workstation software (Agilent Technologies, Santa Clara, CA, USA) was used as the detector. For all compounds, the MS parameters were optimised to obtain the highest signal in MRM mode. The MS was used in dual polarity

CC

mode to detect all compounds in a single run: for all lipophilic vitamin D compounds (D2, D3, 25(OH)D2, 25(OH)D3, 24,25(OH)2D2, 24,25(OH)2D3, 1,25(OH)2D2 and 1,25(OH)2D3), ESI

A

positive ion mode was used and ESI negative mode was used for the four sulfate compounds (D2- S, D3- S, 25(OH)D3- S and 25(OH)D2- S). Deprotonated species ([M−H]−) were selected as precursor ions for all vitamin D-sulfate compounds, whilst for lipophilic vitamin D compounds the protonated precursor ions ([M+H]+) or (M+ H–H2O)+ were used. Table 1 shows the optimised MRM transition parameters, the fragmentor and collision energy (CE) voltages and electrospray ionisation (ESI) mode used. Source parameters including gas flow, 7

sheath gas flow, gas temperature and sheath gas temperature were optimised and maintained at 5 and 12 L/min (gas and sheath gas flows, respectively) and at 300 oC and 250 oC (gas and sheath gas temperatures, respectively). The capillary and nozzle voltages were maintained, respectively, at 5000 V and 2000 V. 2.8.

Method Validation

IP T

The calibration range for each compound was determined based on previously reported concentrations in human serum. The calibration ranges (six levels) were as follows: 0.5 to

100 nM [25(OH)D3, 25(OH)D2, D3, D2, 25(OH)D3-S, 25(OH)D3-S, D2-S, D3-S]; 0.05 to 2 nM

SC R

[1,25(OH)2D3, and 1,25(OH)2D2]; 0.10- 15 nM [24,25(OH)2D3 and 24,25(OH)2D2]. The internal standard concentrations in all calibration standards were: 50 nM [25(OH)D3, 25(OH)D2, D3, D2, 25(OH)D3-S, 25(OH)D3-S, D2-S, D3-S]; 0.5 nM [1,25(OH)2D3, and

1,25(OH)2D2]; 5 nM [24,25(OH)2D3 and 24,25(OH)2D2]. All calibration standards were

U

prepared in mobile phase of the composition at the beginning of the gradient elution (75%B).

N

Linearity was assessed by plotting the peak area ratios of standard/ internal standard against

A

the concentration of the standards using linear regression analysis. The limit of detection (LOD) was calculated as the concentration corresponding to three

M

times the noise, and the limit of quantification (LOQ) as the concentration corresponding to 10 times the noise, for each compound. Repeatability was determined by analysing 10

TE D

separate aliquots of a pooled serum sample followed by the calculation of the percentage relative standard deviation for each vitamin D compound. Recovery was assessed by analysing the concentration of each vitamin D compound in six separate aliquots of spiked

EP

and non-spiked serum samples. Each aliquot of serum was spiked to give a final

CC

concentration similar to the level four of the calibration range used in this study.

A

3.0.

3.1.

Results and discussion LC-MS/MS method development for vitamin D compounds

It is evident from the literature [12] that serum samples are not necessarily subjected to protein precipitation prior to using SPE. We have, however, reported here a simple manual protein precipitation step, to enhance the SPE recovery. The original methodology used in our previous analytical method development involved a protein precipitation step similar to 8

this method, removal of acetonitrile using nitrogen, a subsequent 12 hr freeze drying step followed by the derivatisation of the residual extract before injection onto a column [10]. This lengthy, very labour intensive, sample preparation has now been replaced by a simplified off-line preparation involving manual protein precipitation which is then combined with an on-line SPE pre-treatment, equivalent to a trapping column technique, by using a reusable guard column as the SPE column. The use of a relatively inexpensive guard column as a re-usable SPE cartridge has resulted in diminished operational and consumable costs and

IP T

has significantly enhanced the lifespan of the analytical column. On-line two-dimensional chromatography, using a column-switching device, has been previously shown to reduce

SC R

matrix effects [15].

The optimisation of the on-line clean up and separation was undertaken in two steps: the first stage focused on the extraction of the target compounds using the SPE guard column by

U

injecting the combination standard of vitamin D compounds into the guard column and

N

eluting directly to MS detector; the second stage focused on achieving best resolution of compounds by injecting the combination standard onto the complete assembly using a 1.5 mL

A

sample loop. The MS parameters were optimised by directly infusing the vitamin D standard

TE D

and another ion as the qualifier ion.

M

solutions into the MS detector. The predominant product ion was used as the quantifier ion

There have been previous reports that used a PFP column for underivatised vitamin D compounds [16-18]. As derivatisation was omitted in this study, a PFP column was therefore used in our chromatography. It was also noted that the sensitivities for D-sulfate compounds

EP

were considerably enhanced at higher mobile phase pH values. Therefore, mobile phases was prepared using ammnonium formate or 4-methylmorpholine. The reason for using 4-

CC

methylmorpholine was to prevent any possible adduct formation of some vitamin D compounds with ammonium ions. However, both mobile phases produced similar peak areas

A

for all compounds, confirming that ammonium ions do not appear to form adducts with vitamin D compounds at these conditions. Therefore, ammnonium formate was used as the buffer in the optimised method. The chromatographic separation of 25(OH)D3 from its 3-epi-

25(OH)D3 isomer was necessary to enable quantification of these compounds since they have the same mass transitions. Optimisation of the chromatographic separation using different chromatographic gradients and column temperatures enabled the resolution of these epimers.

9

This agrees with previous studies that have achieved isomeric separation using PFP columns [18-22]. The optimised parameters and their ranges are described in section 2.6. The selected flow rate, mobile phase composition and column temperature obtained are shown in table 2. Chromatograms obtained for a standard mixture, using the optimum LC and MS conditions

Optimisation of protein precipitation method

SC R

3.2.

IP T

are shown in figure 3.

The most efficient protein precipitants for protein removal have previously been reported to be acetonitrile and trichloroacetic acid (TCA) [12]. These precipitants consistently removed plasma proteins effectively in samples from all species examined and at precipitant to plasma

U

volume ratios of 2:1 [12]. In this comparison study, we were not able to detect three

N

compounds, 25(OH)D2, 1,25(OH)2D2 and 24,25(OH)2D2, using either extraction method due to their very low levels in the samples. For all other vitamin D compounds, peak areas

A

observed with TCA precipitation were only 1-6% of the areas observed with acetonitrile

M

precipitation (data not shown). Protein precipitation with acetonitrile is, therefore, seen to be more efficient in releasing vitamin D metabolites from the matrix and/or the acetonitrile

TE D

extract is cleaner and therefore fewer compounds co-elute with the vitamin D peaks resulting in reduced ion suppression in MS detection. In general, it has been shown that improving the sample preparation and the chromatographic separation are the two most effective ways of

Method validation and application to samples

CC

3.3.

EP

circumventing ion suppression [23] in mass spectrometry.

As shown in table 3, the detection and quantification limits were found to be slightly higher,

A

when compared to our previous method [10]. These results may have arisen from our decision to remove the lengthy derivatisation procedure; the derivatisation enhanced the

ionisation to a certain extent and additionally increased the signal/noise ratio since the analytes were detected at higher molecular masses where the noise from low molecular impurities in the mobile phase are reduced. As one of the main objectives of this study was to simplify the method by partially automating the sample preparation thereby making it suitable for routine analysis, we removed the derivatisation step. The lengthy multi-step 10

derivatisation procedure used in vitamin D analysis [10] is not amenable to automation therefore has not been fully or partially automated so far. The sample is required to be completely dry before derivatisation [24], and this is challenging to achieve in an on-line procedure. It has been clearly established that, when co-eluting, isotopically labelled analogues are used as internal standards, there is no significant difference in the results when the calibration

IP T

standards are prepared either in mobile phase solvent or in matrix matched medium [25, 26].

Therefore, all samples and the calibration standards were prepared in mobile phase solvent at

SC R

the same composition as the initial gradient conditions as described in LC-MS/MS section.

The assay calibration demonstrated good linearity (r2 > 0.99) for all 12 vitamin D compounds over the concentration ranges used. We were not able to report the concentrations of three of the vitamin D2 compounds, 1,25(OH)2D2, 25(OH)D2-S and D2-S, due to levels lower than the

U

limit of quantification in the pooled serum used. The method was found to be precise with

N

percentage standard deviations within the range 2.82 to 4.86%. The accuracy of the method, determined as the recovery values, ranged from 92% to 99%. The SD values reported in the

A

recovery column of table 3 gives an approximation of the precision for the three vitamin D2

M

compounds absent in the repeatability column.

TE D

Table 4 shows the ranges of vitamin D compounds in donated human serum samples (n=10) collected from volunteers, using the optimised, validated method reported above. The lowest concentrations of vitamin D compounds were found in volunteers who: had not taken vitamin D supplements; did not use fortified vitamin D milk in their daily intake; and who were rarely

EP

exposed to sunlight in outdoor activities in the past two weeks. The above information was based on surveys provided by the volunteers prior to sample collection. Most experts agree

CC

that a 25(OH)D3 concentration below 20 ng/mL (50 nmol/L) [27-30] is an indication of vitamin D deficiency, whereas concentrations of 21–29 ng/mL (51–74 nmol/L) are

A

considered to indicate insufficiency and concentrations of 30 ng/mL (75 nmol/L) suggested by some to maximise the effect of vitamin D on calcium, bone, and muscle metabolism [3032]. Severe deficiency leads to rickets in children and osteomalacia in adults with concentrations of 25(OH)D3 below 10 ng/mL (25 nmol/L). The chromatographic profiles obtained for human serum are shown in figure 4. The broadness of some peaks may likely to be due to incomplete separation of epimeric forms. The only epimeric form separated and identified in this study, using standard compound, is the epi-25(OH)D3. 11

4.0.

Conclusions

This paper is the first to report the development and validation of an analytical method for the simultaneous detection and quantification of 12 vitamin D compounds (both lipophilic and hydrophilic) in human serum. The high accuracy, selectivity and sensitivity obtained

IP T

demonstrate the suitability of the proposed method for the determination of vitamin D

compounds and their metabolites in man, that are potentially useful in disease diagnosis. The

method described herein is fast, cost-effective, and has minimum sample handling; making it

SC R

suitable for routine analysis. The method can be very likely applied to any biological fluid

Acknowledgements

A

N

5.0.

U

following modification of the sample preparation step.

The authors wish to thank the volunteers who donated samples. Nur Sofiah Abu Kassim is

A

CC

EP

TE D

M

sponsored by the Ministry of Education and Universiti Teknologi MARA, Malaysia.

12

6.0.

References

A

CC

EP

TE D

M

A

N

U

SC R

IP T

[1] M.J. Müller, D.A. Volmer, Mass Spectrometric Profiling of Vitamin D Metabolites beyond 25‐ Hydroxyvitamin D, Clin. Chem., 61 (2015) 1033‐1048.  [2] J.M. van den Ouweland, Analysis of vitamin D metabolites by liquid chromatography‐tandem  mass spectrometry, Trends Anal. Chem., 84 (2016) 117‐130.  [3] D.A. Volmer, Analysis of vitamin D metabolic markers by mass spectrometry: Current techniques,  limitations of the “gold standard” method, and anticipated future directions., Mass Spectrom. Rev.,  34 (2015) 2‐23.  [4] M.F. Holick, Vitamin D status: measurement, interpretation, and clinical application, Ann.  Epidemiol., 19 (2009) 73‐78.  [5] M.J. Berridge, Vitamin D deficiency and diabetes, Biochem. J., 474 (2017) 1321‐1332.  [6] R.M. Daly, C. Gagnon, Z.X. Lu, D.J. Magliano, D.W. Dunstan, K.A. Sikaris, P.Z. Zimmet, P.R. Ebeling,  J.E. Shaw, Prevalence of vitamin D deficiency and its determinants in Australian adults aged 25 years  and older: a national, population‐based study, Clin. Endocrinol., 77 (2012) 26‐35.  [7] F.P. Gomes, Shaw P Nicholas, Karen Whitfield, Pieter Koorts, Amitha K Hewavitharana, Recent  trends in the determination of vitamin D, Bioanalysis, 5 (2013) 3063‐3078.  [8] M.J. Müller, C.S. Stokes, D.A. Volmer, Quantification of the 3α and 3β epimers of 25‐ hydroxyvitamin D3 in dried blood spots by LC‐MS/MS using artificial whole blood calibration and  chemical derivatization, Talanta, 165 (2017) 398‐404.  [9] T. Higashi, A. Goto, M. Morohashi, S. Ogawa, K. Komatsu, T. Sugiura, T. Fukuoka, K. Mitamura,  Development and validation of a method for determination of plasma 25‐hydroxyvitamin D3 ‐3‐ sulfate using liquid chromatography/tandem mass spectrometry, J. Chromatogr. B. Analyt. Technol.  Biomed. Life. Sci., 969 (2014) 230‐234.  [10] N.S. Abu Kassim, F.P. Gomes, P.N. Shaw, A.K. Hewavitharana, Simultaneous quantitative analysis  of nine vitamin D compounds in human blood using LC‐MS/MS, Bioanalysis, 8 (2016) 397‐411.  [11] F.P. Gomes, P.N. Shaw, A.K. Hewavitharana, Determination of four sulfated vitamin D  compounds in human biological fluids by liquid chromatography‐tandem mass spectrometry, J.  Chromatogr. B. Analyt. Technol Biomed Life Sci., 1009‐1010 (2015) 80‐86.  [12] C. Polson, P. Sarkar, B. Incledon, V. Raguvaran, R. Grant, Optimization of protein precipitation  based upon effectiveness of protein removal and ionization effect in liquid chromatography–tandem  mass spectrometry, J. Chromatogr. B. Analyt. Technol Biomed Life Sci., 785 (2003) 263‐275.  [13] D. Thibeault, N. Caron, R. Djiana, R. Kremer, D. Blank, Development and optimization of  simplified LC‐MS/MS quantification of 25‐hydroxyvitamin D using protein precipitation combined  with on‐line solid phase extraction (SPE), J. Chromatogr. B. Analyt. Technol Biomed Life Sci., 883‐884  (2012) 120‐127.  [14] F.P. Gomes, P.N. Shaw, K. Whitfield, A.K. Hewavitharana, Simultaneous quantitative analysis of  eight vitamin D analogues in milk using liquid chromatography‐tandem mass spectrometry, Anal.  Chim. Acta, 891 (2015) 211‐220.  [15] R. Pascoe, J.P. Foley, A.I. Gusev, Reduction in matrix‐related signal suppression effects in  electrospray ionization mass spectrometry using on‐line two‐dimensional liquid chromatography,  Anal. Chem., 73 (2001) 6014‐6023.  [16] S. Baecher, A. Leinenbach, J.A. Wright, S. Pongratz, U. Kobold, R. Thiele, Simultaneous  quantification of four vitamin D metabolites in human serum using high performance liquid  chromatography tandem mass spectrometry for vitamin D profiling, Clin. Biochem, 45 (2012) 1491‐ 1496.  [17] R.L. Schleicher, S.E. Encisco, M. Chaudhary‐Webb, E. Paliakov, L.F. McCoy, C.M. Pfeiffer, Isotope  dilution ultra performance liquid chromatography‐tandem mass spectrometry method for  simultaneous measurement of 25‐hydroxyvitamin D2, 25‐hydroxyvitamin D3 and 3‐epi‐25‐

13

A

CC

EP

TE D

M

A

N

U

SC R

IP T

hydroxyvitamin D3 in human serum, Clin. Chim. Acta; international journal of clinical chemistry, 412  (2011) 1594‐1599.  [18] J.M. van den Ouweland, A.M. Beijers, H. van Daal, Fast separation of 25‐hydroxyvitamin D3 from  3‐epi‐25‐hydroxyvitamin D3 in human serum by liquid chromatography–tandem mass spectrometry:  variable prevalence of 3‐epi‐25‐hydroxyvitamin D3 in infants, children, and adults, Clin. Chem., 57  (2011) 1618‐1619.  [19] D. Bailey, K. Veljkovic, M. Yazdanpanah, K. Adeli, Analytical measurement and clinical relevance  of vitamin D3 C3‐epimer, Clin. Biochem, 46 (2013) 190‐196.  [20] R.J. Singh, R.L. Taylor, G.S. Reddy, S.K. Grebe, C‐3 epimers can account for a significant  proportion of total circulating 25‐hydroxyvitamin D in infants, complicating accurate measurement  and interpretation of vitamin D status, J. Clin. Endocrinol Metab., 91 (2006) 3055‐3061.  [21] F.G. Strathmann, K. Sadilkova, T.J. Laha, S.E. LeSourd, J.A. Bornhorst, A.N. Hoofnagle, R. Jack, 3‐ epi‐25 hydroxyvitamin D concentrations are not correlated with age in a cohort of infants and adults,  Clin. Chim. Acta, 413 (2012) 203‐206.  [22] A. Mena‐Bravo, F. Priego‐Capote, M.D.L. de Castro, Two‐dimensional liquid chromatography  coupled to tandem mass spectrometry for vitamin D metabolite profiling including the C3‐epimer‐ 25‐monohydroxyvitamin D‐3, J. Chromatogr. A., 1451 (2016) 50‐57.  [23] L.L. Jessome, D.A. Volmer, Ion suppression: a major concern in mass spectrometry, LC GC N.  Am., 24 (2006) 498.  [24] P.A. Aronov, L.M. Hall, K. Dettmer, C.B. Stephensen, B.D. Hammock, Metabolic profiling of major  vitamin D metabolites using Diels–Alder derivatization and ultra‐performance liquid  chromatography–tandem mass spectrometry, Anal. Bioanal. Chem., 391 (2008) 1917‐1930.  [25] A.K. Hewavitharana, Matrix matching in liquid chromatography–mass spectrometry with stable  isotope labelled internal standards—Is it necessary?, J. Chromatogr. A., 1218 (2011) 359‐361.  [26] X.‐D. Pan, W. Jiang, P.‐G. Wu, Comparison of different calibration approaches for  chloramphenicol quantification in chicken muscle by ultra‐high pressure liquid chromatography  tandem mass spectrometry, Analyst, 140 (2015) 366‐370.  [27] M.F. Holick, High prevalence of vitamin D inadequacy and implications for health, in:  Mayo  Clinic Proceedings, Elsevier, 2006, pp. 353‐373.  [28] H.A. Bischoff‐Ferrari, E. Giovannucci, W.C. Willett, T. Dietrich, B. Dawson‐Hughes, Estimation of  optimal serum concentrations of 25‐hydroxyvitamin D for multiple health outcomes, Am. J. Clin.  Nutr., 84 (2006) 18‐28.  [29] A. Malabanan, I. Veronikis, M. Holick, Redefining vitamin D insufficiency, Lancet, 351 (1998)  805‐806.  [30] Ri, Hypovitaminosis in medical inpatients, N. Engl. J. Med, 338 (1998) 777.  [31] M.C. Chapuy, P. Preziosi, M. Maamer, S. Arnaud, P. Galan, S. Hercberg, P.J. Meunier, Prevalence  of vitamin D insufficiency in an adult normal population, Osteoporos Int., 7 (1997) 439‐443.  [32] M.F. Holick, E.S. Siris, N. Binkley, M.K. Beard, A. Khan, J.T. Katzer, R.A. Petruschke, E. Chen, A.E.  de Papp, Prevalence of Vitamin D inadequacy among postmenopausal North American women  receiving osteoporosis therapy, J. Clin. Endocrinol. Metab., 90 (2005) 3215‐3224. 

14

7.0.

Figure Captions

N

U

SC R

IP T

Figure 1. Chemical structures of the deuterated analogues of vitamin D compounds used in the analysis

A

CC

EP

TE D

M

A

Figure 2. Scheme of the two valve positions; Flow shown in black

Figure 3. LC–MS/MS profiles of 12 vitamin D compounds in the standard mixture

15

IP T SC R

A

CC

EP

TE D

M

A

N

U

Figure 4. Chromatographic profiles showing all detectable vitamin D compounds in human serum samples

16

Table 1. Optimised mass spectrometry parameters used in MS detection MRM transition Vitamin D compounds

Fragmentor

Collision energy

MRM mode

Qualifier ion (m/z)

(V)

(V)

162

104

24

Positive

D3-d3

388 - 162

---

104

24

Positive

D2

397 - 159

---

100

43

Positive

D2-d3 Epi-25(OH)D3 Epi-25(OH)D3-d3 25(OH)D3

400 - 162 401 - 159 407 - 165 401 - 159

--257 --257

100 100 100 100

25(OH)D3-d6

407 - 165

---

25(OH)D2

413 - 159

---

25(OH)D2-d6

419 - 165

---

25(OH)D3-S

479 - 96

---

25(OH)D3-S-d3

482 – 96

---

25(OH)D2-S

491 - 96

25(OH)D2-S-d3

494 - 96

1,25(OH)2D3

417 - 399

1,25(OH)2D3-d6 D3-S

Positive Positive Positive Positive

100

28

Positive

90

24

Positive

90

24

Positive

222

36

Negative

222

36

Negative

---

222

40

Negative

---

222

40

Negative

381

124

4

Positive

423 - 405

---

124

4

Positive

463 - 96

---

232

36

Negative

466 - 96

---

232

36

Negative

411 - 151

133

144

4

Positive

417 - 157

---

144

4

Positive

475 - 96

---

242

36

Negative

478 - 96

---

242

36

Negative

24,25(OH)2D3

417 - 381

159

90

12

Positive

24,25(OH)2D3-d3

420 - 384

---

90

12

Positive

24,25(OH)2D2

429 - 393

111

60

4

Positive

24,25(OH)2D2-d6

435 - 399

---

60

4

Positive

D3-S-d3 1,25(OH)2D2 –H2O 1,25(OH)2D2-d6

U

N A

M

A

CC

D2-S-d3

EP

D2-S

SC R

43 28 28 28

TE D

D3

IP T

Precursor ion Product ion (m/z) 385 - 159

17

Table 2. Optimum condition of the automated SPE step Optimum condition

Variable

Pentafluorophenyl (PFP) 0.8 mL/ min Ammnonium formate 40:60 (water:methanol) 40˚C 6.17 (3 mM ammnonium formate)

Pentafluorophenyl (PFP) 0.2 mL/ min Ammnonium formate 25: 75 (water:methanol) 40˚C 6.17 (3 mM ammnonium formate)

A

CC

EP

TE D

M

A

N

U

SC R

Temperature pH

PFP column

IP T

Type Flow rate Buffer Mobile phase composition

Guard Column

18

I N U SC R

Table 3. Linearity, repeatability, recovery, limit of detection (LOD) and limit of quantification (LOQ) of all vitamin D compounds including epi25(OH)D3 determined using a pooled serum sample.

A

Equation

Vitamin D compounds

Repeatability (n=10)

M

r2

0.9974 0.9957 0.999 0.9939 0.9968 0.9902 0.9905 0.9976 0.9978 0.9904 0.9934 0.9997 0.9999

Mean ± SD (nmol/L) 51.50 ± 1.48 2.1200 ± 0.0686 10.800 ± 0.341 14.500 ± 0.627 2.230 ± 0.109 0.6240 ± 0.0233 N.D 1.1200 ± 0.0357 3.370 ± 0.120 12.000 ± 0.529 N.D 1.2000 ± 0.0338 N.D

A

CC E

PT

ED

25(OH)D3 y= 13.551x + 0.0314 epi-25(OH)D3 y= 4.8937x - 0.1135 25(OH)D2 y= 0.6559x + 0.2549 D3 y= 0.0668x + 0.3179 D2 y= 1.7506x - 2.1213 1,25(OH)2D3 y= 0.151x + 0.018 1,25(OH)2D2 y= 2.1163x + 0.932 24,25(OH)D3 y= 1.9274x - 0.966 24,25(OH)D2 y= 5.6412x + 0.9608 25(OH)D3-S y= 0.0076x + 0.1845 25(OH)D2-S y= 0.1641x + 0.9365 D3-S y= 1.524x - 0.0496 D2-S y= 63.194x + 3.4594 N.D, Not detected; SD, standard deviation

19

RSD % 2.87 3.23 3.17 4.33 4.86 3.57 N.D 3.18 3.6 4.41 N.D 2.82 N.D

%Recovery (n=6) Mean ± SD (nmol/L) 97.5 ± 1.61 97.9 ± 0.827 97.8 ± 4.18 92.5 ± 0.291 92.2 ± 1.26 93.3 ± 0.0230 94.9 ± 0.0490 93.2 ± 2.88 96.1 ± 0.0430 97.8 ± 0.667 95.7 ± 1.35 99.7 ± 0 1.32 97.3± 0.0690

LOD (LOQ) [nmol/L) 0.037 (0.122) 0.037 (0.122) 0.012 (0.0402) 0.019 (0.0628) 0.048 (0.158) 0.024 (0.0796) 0.068 (0.227) 0.018 (0.0598) 0.025 (0.0831) 0.059 (0.1950) 0.182 (0.606) 0.026 (0.0849) 0.033 (0.109)

Concentration ranges (nmol/L) 18.7 - 83.2 6.43 - 47.0 0.0654 - 14.0 2.63 - 18.3 0.00 - 3.65 0.263 - 1.08 0.00 - 2.62 0.264 - 3.04 0.00 - 0.159 9.52 - 43.8 0.00 - 40.0 0.0976 - 2.75 0.00 - 0.363

A

CC

EP

TE D

M

A

N

U

SC R

Vitamin D compounds 25(OH)D3 epi-25(OH)D3 25(OH)D2 D3 D2 1,25(OH)2D3 1,25(OH)2D2 24,25(OH)2D3 24,25(OH)2D2 25(OH)D3-S 25(OH)D2-S D3-S D2-S

IP T

Table 4. Concentration ranges of vitamin D compounds in serum from ten volunteers

20