Eliot Chung

Vitamin D comprises a group of secosteroids, and mainly refers to two physiologically inactive fatsoluble prohormones: vitamin D

 is photochemically synthesized from 7dehydrocholesterol in the epidermal layer of the skin of vertebrates under ultraviolet (UV) B light (270–290 nm) radiation. Vitamin D

 is derived from fungal and plant sterol when ergosterol is exposed to UV radiation. Vitamin D

 is not produced by the human body and is much less effective than vitamin D

 is hydroxylated in the liver and converted to a nonactive storage form of 25hydroxycholecalciferol, which is further metabolized in the kidneys into the physiologically major active hormone, 1,25dihydroxycholecalciferol. This hormone is the main circulating form of vitamin D, which enhances the ability of the small intestine to absorb calcium and retain phosphate from the diet, and prevents metabolic bone diseases such as rickets, osteomalacia, osteoporosis and agerelated macular degeneration,

  which are caused by vitamin D deficiency. 

In 2007, the Canadian Cancer Society recommended that adults living in Canada should consider taking vitamin D supplements of 1000 IU/day during the autumn and winter, and those at higher risk of lower vitamin D levels should take 1000 IU/day all year round.

  Meanwhile, the US Dietary Reference Intake for Adequate Intake of vitamin D

 lists: 200 IU/day as adequate for children and adults aged ≤50 years, 400 IU/day for adults aged 51–70 years, and 600 IU/day for adults aged >71 years.

Fifteen experts from universities, research institutes and university hospitals worldwide called for international agencies, such as the Food and Nutrition Board (WA, USA) and the European Commission's Health and Consumer Protection DirectorateGeneral (Belgium), to reassess dietary recommendations for vitamin D as a matter of high priority because the formal advice from health agencies is outdated and putting the public at risk.

  Currently, the FDAapproved prescription oral tablet drug FOSAMAX PLUS D (70 mg alendronate sodium/5600 IU cholecalciferol) has been widely used for people diagnosed with osteoporosis. Alendronate slows bone loss and increases bone mass, which helps prevent bone fractures, while cholecalciferol aids bone absorption of calcium.  

, are usually clinical indicators of nutritional vitamin D deficiency in humans.

  Based on FDA guidance for bioavailability and bioequivalence studies for orally administered drug products, the moieties to be measured in biological fluids collected are either the API or its active moiety in the administered dosage form (parent drug) and, when appropriate, its active metabolites.  

For bioequivalence studies, measurement of the concentration–time profile of the parent drug released from the dosage form, rather than the metabolite, is generally recommended because it is more sensitive to changes in formulation performance than metabolites.  

Measurement of a metabolite may be preferred when parent drug levels are too low to allow reliable analytical measurement in blood, plasma or serum for an adequate length of time, or when a metabolite may be formed as a result of gut wall or other presystemic metabolism.

  Therefore, it is of more clinical importance to directly monitor vitamin D

 levels in the human body when a vitamin D drug or a supplement is taken.  

Previous determination of endogenous vitamin D has mainly been conducted by high performance liquid chromatography/ultraviolet (HPLC/UV) and gas chromatography/mass spectrometry

(GC/MS) methods. These methods were considered challenging because their low sensitivities for low vitamin D levels in circulation required highblood volumes. The similarity between vitamin D and its analogs and metabolites meant that HPLC methods suffered from inadequate chromatographic separation and lacked specificity of interference. The dynamic endogenous concentration of vitamin D in the presence of UV light, and complex labourintensive and timeconsuming sample extraction procedures from ligand binding assays and low recovery from derivatization methods were also problematic.

LC–MS/MS methods have been widely used for the determination of vitamin D

 metabolites and analogs, but not very much for the direct determination of endogenous vitamin D

This paper presents an atmosphericpressure chemical ionization (APCI)LC–MS/MS method developed and validated to accurately measure the absolute quantities of endogenous vitamin D

Cholecalciferol was purchased from Sigma Aldrich (Canada) and hexadeuterated (d

 was purchased from Chemphor Inc. (Canada). Blank human plasma with dipotassium ethylenediaminetetraacetic acid (EDTA) anticoagulant was received frozen on dry ice from Bioreclamation Inc. (NY, USA). Methanol (HPLC grade) and acetonitrile (Omnisolv grade) were from EMD (NJ, USA). Formic acid (ACS grade) was from Fisher Scientific (Canada) and deionized water was prepared in a Millipore (France) water system (MilliQ Gradient Ultrapure).  

An MDS/Sciex API5000 triple quadrupole mass spectrometer (MDS Sciex, Canada) interfaced with an APCI source (MDS Sciex) coupled with an Agilent 1100 series thermostatted column compartment (Agilent Technologies, Switzerland), a binary pump (Agilent Technologies), a degasser (Agilent Technologies), a Leap Technologies' (Switzerland) CTC analytics LC PAL series autosampler, and a VICI (TX, USA) valco valve, was used and controlled by Analyst (Applied Biosystems, MA, USA). 

LC was conducted on a Zorbax Eclipse XDBC8 column (Agilent Technologies, USA) (50 × 4.6 mm, 3.5μm) maintained at 25 °C. A total of 20 μL of each sample was injected onto the column. The mobile phase operated at a flow rate of 1200 μL/min and consisted of 0.1% (v/v) formic acid in water as solvent A and methanol as solvent B under gradient elution.  

, were prepared in methanol and stored at –40 °C. A sufficient amount of frozen inhouse prepared surrogate human plasma was thawed.  

Quality control samples at 0.25 ng/mL (QC-LOQ), 0.75 ng/mL (QC-L), 6.00 ng/mL (QC-M1), 12.50 ng/mL (QCM2) and 18.00 ng/mL (QC-H) were prepared in surrogate human plasma, aliquoted in small volume vials and stored at –40 and –70°C for stability assessment.  

Calibration standards with a range of 0.25–25.00 ng/mL were freshly prepared daily in surrogate human plasma. Vitamin D

 samples (200 μL aliquots) were prepared using a protein precipitation method. After precipitation, the supernatant layer was transferred, evaporated to dryness under nitrogen gas and the residue was redissolved with 150 mL of methanol:water mixture. 

 is a white crystalline powder soluble in 100% ethanol, chloroform or acetone and insoluble in water. It is light sensitive and should be protected from exposure to light. The stable isotopelabelled d

 was used as an internal standard for quantitation of vitamin D

 (Figure 1). The electrospray ionization source (ESI) showed very low sensitivity to vitamin D

 and its internal standard because of the lack of protonation sites on the fatsoluble vitamins, making their ionization very difficult. The relatively stable structure of vitamin D

Figure 1: Structures of vitamin D3 and its internal standard, d6-vitamin D3. 

APCI, previously found to be a suitable ionization source for the characterization of steroids, was used for vitamin D

  Although the product ions of m/z 367.2 from vitamin D

 show relatively stronger signal intensity than those of m/z 259.2 and m/z 265.2, the former were not used because they are the fragment ions of the parent molecular ions after losing one water molecule ([M + H-H

Significant matrix interference was also observed in later chromatographic separation when using m/z 367.2. Optimal multiple reaction monitoring (MRM) transitions at m/z 385.2–259.2 for vitamin D

 is an endogenous component in human plasma reported in the range 0.7–5.7 ng/mL

 in humans is dynamically changed daily, with its skin biosynthesis affected by multiple factors, such as altitude, sun exposure, clothing, season, ageing and skin melanin pigmentation. 

FDA recommends that the same biological matrix as the samples in the intended study be used for the standard calibration curve, prepared by spiking the matrix with known concentrations and that the low limit of quantitation (LLOQ) response should be at least five times the response of a blank.

  However, it is impractical to prepare the standard calibration curves and quality control samples in human plasma because of the endogenous vitamin D

Proper selection of a surrogate matrix to establish a quantitative standard calibration curve and quality control samples for vitamin D

 for the range 0.25–25.00 ng/mL posed enormous challenges for the establishment of an effective bioanalytical method for the determination of endogenous vitamin D

 in human plasma. Different approaches in the selection of a surrogate matrix for determining endogenous components in biological matrices have been reported, such as preparation of calibration standards of seven endogenous ketolic estrogens in methanol instead of human urine;

  pretreatment of blank human nerve tissues instead of using normal human nerve tissue for determination of endogenous sorbitol and fructose;

  preparation of endogenous hormones in solutions of phosphate buffer and bovine serum albumin instead of human serum and urine to prevent contamination from various endogenous hormones;

  determination of endogenous estrogens using charcoalstripped human urine;

  and calculation of endogenous components using background substraction.  

Various lots of charcoalstripped human plasma from different commercial sources were tested and evaluated and significant amounts of vitamin D

 were still observed in these custommade blank human plasma samples. Exposing the charcoalstripped blank human plasma samples directly to indoor light for 48 h did not reduce endogenous vitamin D

Several other surrogate matrices, such as human serum, rat plasma and serum, mouse plasma and serum, and human and bovine serum albumin were also evaluated for the assay.

  The different surrogate matrices also affected vitamin D

An inhouse method developed to strip vitamin D

 from commercial blank human plasma using a charcoalstripping technique was successfully established. The charcoal particle size distribution, surface area, porosity, carbon percentage, and the treatment method of the activated charcoal were the keys to the successful preparation of quality vitamin D

For standards prepared in surrogate matrix 2 (Figure 2), the slope of the standard calibration curve was significantly lower for the extracted samples stored overnight at –40 °C than that of the extracted samples that were freshly prepared.    

Figure 2: Effect of surrogate matrix on vitamin D3 standard calibration curves. 

For surrogate matrix 1 (Figure 2), the slope of the standard calibration curve shows no significant difference between samples that were freshly prepared or frozen. Supplementary data (

) show a chromatogram of endogenous vitamin D

 from commercial blank human plasma. No vitamin D

 was observed at the retention time of vitamin D

 in the selected surrogate human plasma.  

 is a highly hydrophobic compound that strongly retains onto the chromatographic column. Various brands and types of reversephase columns were tested and it was found that vitamin D

 could not be eluted from all tested C18 columns when using 100% methanol or acetonitrile for 15 min.  

Reasonable retention time and good peak shape were achieved when using Synergy 4 μm Polar-RP 80 A° (50×4.6 mm, 4μm) (Phenomenex, USA) and Zorbax Eclipse XDBC8 (50×4.6 mm, 3.5μm) (Agilent Technologies, USA) columns. However, the vitamin D

 peak overlapped with the shoulder of the significant interference peak of an endogenous unknown when using the former column.  

Two major interference peaks with retention times at 2.0–2.4 and 2.8–3.5 min were observed before and after the vitamin D

 peak (Figure 3a). Complete baseline separation of vitamin D

 from the endogenous interference peaks was obtained after further optimization of the LC elution gradient (Figure 3b).    

Figure 3: Optimization of LC gradient to separate vitamin D3 from interferences. 

 spiked in surrogate human plasma at LLOQ of 0.25 ng/mL after optimization of the LC gradient. Vitamin D

 was well separated from all other interference peaks appearing between 1.0–1.5 min. The major interference peak at 4.5 min is not shown in the chromatogram as the elution solution was switched to waste after 2.8 min to reduce introduction of matrix interference into the LC–MS/MS system.  

) show a representative chromatogram of spiked internal standard with the same retention time as vitamin D

, but without any interference peaks shown in the detection MRM channel. 

The poor water solubility and strong binding ability to globulin proteins in human blood make vitamin D

 very difficult to extract. Liquid–liquid extraction and protein precipitation methods were both tested for vitamin D

 sample preparation and found to obtain similar results.  

The less labourintensive and costeffective protein precipitation method was used for sample preparation. To avoid vitamin D

 coprecipitation and improve extraction recovery, it is recommended to thoroughly mix and vortex before centrifugation.  

The influence of the containers used for vitamin D

 preparation and storage was also investigated. Although no significant difference was observed for standard calibration curves prepared in either plastic or glass vials, the accuracy of QC-L, QC-M1 and 5-fold diluted samples prepared in glass vials was out of the acceptance criteria for QC samples (Figure 4).    

Figure 4: Effects of vitamin D3 adsorption on (a) standard and (b) accuracy of quality control. 

Articles

An LC&#150;MS/MS method for the quantitative determination of vitamin D3 in human plasma has been developed and validated with positive atmospheric chemical ionization sources.