Journal of Chromatography B (v.991, #C)

Biotransformation of luteoloside by a newly isolated human intestinal bacterium using UHPLC-Q-TOF/MS by Jin-hua Tao; Dong-geng Wang; Chi Yang; Jin-hua Huang; Wen-qian Qiu; Xi Zhao (1-8).
To explore the metabolic pathways and metabolites of luteoloside yielded by the isolated human intestinal bacteria from healthy human feces and characterize the β-d-glucosidase activity of the specific strain which catalyzed the breakdown of luteoloside, a preculture bacterial GAM broth and luteoloside were mixed incubated together for 48 h. UHPLC-Q-TOF/MS was used for analysis of the metabolites of luteoloside in the corresponding supernatant fractions from fermentation. Aliquots of the reactive solutions were collected at different times and were measured with a microplate reader at 405 nm to evaluate the enzymatic activity. Three metabolites (acetylated luteoloside, luteolin and deoxygenated luteolin) were detected in the fractions isolated from the bacterial samples. The variation of β-d-glucosidase activity inside the bacterium was in coincidence with the changes in luteolin generation or luteoloside degradation in different time periods.
Keywords: Luteoloside; Enterococcus sp. 45; Metabolic pathway; UPLC-Q-TOF/MS; Enzyme activity;

In this study, a method for the quantitation of SK5474 in rat plasma was developed and validated using high performance liquid chromatography (HPLC). The plasma samples were prepared by deproteinization, and sildenafil was used as an internal standard. Chromatographic separation was achieved using a reversed-phase (C18) column. The mobile phase, 0.02 M ammonium acetate buffer:acetonitrile (48:52, v/v), was run at a flow rate of 1.0 mL/min, and the column eluent was monitored using an ultraviolet detector at 254 nm at room temperature. The retention times of sildenafil and SK5474 were approximately 5.8 and 7.2 min, respectively. The quantitation limit of SK5474 in rat plasma was 0.03 μg/mL. Pharmacokinetic parameters of SK5474 was evaluated after intravenous (i.v.; at doses of 15 mg/kg) and oral (p.o.; at doses of 30 mg/kg) administration of SK5474 in rats. After p.o. administration (30 mg/kg) of SK5474, F (fraction absorbed) value was approximately 46.0%.
Keywords: SK5474; Sildenafil; HPLC; Bioavailability; Rats;

Pharmacokinetics and tissue distribution study of Isovitexin in rats by HPLC-MS/MS by Yaxin Li; Yanqing Zhang; Tan Yang; Hui Li; Jiang Guo; Qiqing Zhao; Junbo Xie (13-20).
A sensitive and credible high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established and validated for the determination of isovitexin in rat plasma and various tissues (including heart, liver, lung, kidney, stomach, intestine, muscle, brain and cerebellum). The samples were prepared with methanol by liquid–liquid extraction, and puerarin was used as the internal standard. The chromatographic separation was carried out on an Agilent Poroshell 120 EC-C18 column (4.6 mm × 50 mm, 2.7 μm) with a mobile phase consisting of acetonitrile and 0.1% formic acid (21:79, v/v). The MS analysis was performed by multiple reaction monitoring (MRM) with electronic spray ionization source (ESI) for quantitative response of isovitexin (431.0→311.0) and puerarin (415.1→295.0). The linearity of isovitexin in all the biosamples was good, with correlation coefficients greater than 0.9912 within the corresponding concentration range. The intra- and inter-day precisions in plasma and various tissues were less than 11.80%, and the accuracy (RE %) ranged from −4.89% to 4.78%. The extraction recoveries were in the range of 72.70%–90.81%. The present method was successfully applied to pharmacokinetics and tissue distribution of isovitexin in rats after tail vein injection with 2.0 mg/kg of the compound. The pharmacokinetic parameters were demonstrated as followed: the half-life (t 1/2) was 1.05 ± 0.325 h, the apparent volume of mean residual time (MRT) was 1.229 ± 0.429 h, and the area under the curve (AUC) was 11.39 ± 5.05 μg/mL/h. The results of tissue distribution showed that the main tissue depots for isovitexin in rats were kidney, intestine and liver. The results provided a meaningful insight for the further pharmacological investigation of isovitexin.
Keywords: Isovitexin; HPLC-MS/MS; Pharmacokinetics; Tissue distribution;

Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography by Mindaugas Zaveckas; Simas Snipaitis; Henrikas Pesliakas; Juozas Nainys; Alma Gedvilaite (21-28).
Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impact on swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccine candidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizes were examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. Q Sepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some host cell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6B and CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPs after chromatography on Heparin Sepharose CL-6B was only 4–7% and the recovery of VLPs was 5–7%. Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purity of about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 monolith was 15–18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electron microscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate was developed using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith.
Keywords: Porcine circovirus type 2; Virus-like particles; Monolith; Ion-exchange chromatography;

A simple U-HPLC-MS/MS method for the determination of liensinine and isoliensinine in rat plasma by Li-Sheng Peng; Xiao-Yan Jiang; Zhong-Xin Li; Tie-Gang Yi; Bin Huang; Hui-Lin Li; Zhen Zeng; Yu Liu; Song-Ling Peng; Jin-Song He; Li He; Li-Ping Peng (29-33).
An ultra-high performance liquid chromatography tandem mass spectrometry (U-HPLC-MS/MS) method was developed and validated to determine liensinine and isoliensinine in rat plasma simultaneously. Plasma samples were prepared using protein precipitation with acetonitrile. The two analytes and the internal standard pirfenidone were separated on an Acquity U-HPLC BEH C18 column with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. Both liensinine and isoliensinine were eluted at 0.63 and 0.82 min, respectively. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 611.6 → 206.2 for liensinine and m/z 611.4 → 192.2 for isoliensinine. The linearity of this method was found to be within the concentration range of 5–700 ng/mL for liensinine and isoliensinine in rat plasma. The lower limits of quantification (LLOQ) were all 5 ng/mL for liensinine and isoliensinine. The relative standard deviations (RSD) of intra and inter precision were less than 10% for both liensinine and isoliensinine. The method was also successfully applied to the pharmacokinetic study of liensinine and isoliensinine in rats.
Keywords: Liensinine; Isoliensinine; U-HPLC-MS/MS; Rat plasma; Pharmacokinetic;

Midodrine (MD) is a prodrug that is converted after oral administration to Desglymidodrine (DMD). In this study, an LC–MS/MS assay was developed and validated for investigation of the pharmacokinetics of MD and DMD in non azotemic patients with liver cirrhosis and tense ascites. Results were compared to those noted with healthy volunteers following the adminstration of a single oral dose of MD. Sample preparation was performed by liquid–liquid extraction using t-butyl methyl ether. HPLC separation was carried out using RP C18 column (4.6 mm × 50 mm, 5 μm). Isocratic elution was performed using methanol:0.2% formic acid (70:30, v/v) as the mobile phase, at a flow rate of 0.7 mL/min. Tandem mass spectrometric detection was employed at positive electrospray ionization in MRM mode for the determination of MD and DMD. Analysis was carried out within 1.0 min over a concentration range of 0.50–40.00 ng/mL for the prodrug and its active metabolite. The assay was validated according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the determination of the pharmacokinetic parameters of MD and DMD and personalized therapy was demonstrated in healthy volunteers and ascitic patients. Results revealed significant differences in pharmacokinetic parameters among the studied groups. Such differences were explained on the basis of the medical condition and co-adminstered medications exerting possible drug–drug interaction. Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment.
Keywords: Individualized therapy; Comparative pharmacokinetics; LC–MS/MS; Midodrine; Desglymidodrine; Ascites;

Unraveling the constituents of biological samples using HPLC is a central core technology in metabolomics experiments. Consistency in retention time across many samples is a critical criterion for judging the quality of a data set, which must be met before further analysis are possible. Here, the performance of two ultra high-performance liquid chromatography (UHPLC) systems has been compared using an established separation protocol optimized for phenylpropanoids, a class of secondary compounds found in plants displaying intermediate polarity. The two systems differed markedly with respect to their reproducibility and pressure stability. The standard deviation of the retention time of representative peaks differs up to 30-folds between the systems. Adjustments made to the gradient profiles succeeded in equalizing their level of performance. However, the modifications made to the separation protocol reduced the quality of the separation, particularly of the more rapidly eluting components, and lengthened the run time.
Keywords: U(HPLC); Retention time; MS based detection; Secondary metabolites; Metabolomic profiling;

Evaluation of plasma microsampling for dried plasma spots (DPS) in quantitative LC-MS/MS bioanalysis using ritonavir as a model compound by Wenkui Li; John Doherty; Sarah Favara; Christopher Breen; Jimmy Flarakos; Francis L.S. Tse (46-52).
Quantitative bioanalysis of dried plasma spots (DPS) is not subject to the impact of hematocrit and sample non-homogeneity that are often encountered in dried blood spot (DBS) assay. In the present report, an evaluation of plasma microsampling for DPS has been conducted for the first time using ritonavir as a model compound orally administered to dogs. For this evaluation, an LC-MS/MS method was developed and validated according to the current health authorities’ guidance and industry practice for the analysis of ritonavir in DPS samples. The measured ritonavir concentrations in the DPS samples prepared using SAFE-TEC devices and directly from the conventional wet plasma using standard pipette were compared with each other and against those of conventional wet plasma. Both DPS results correlated well with each other and were comparable to those of the wet plasma. Good incurred sample reanalysis results were obtained for the two sets of DPS samples and wet plasma as well. The current plasma microsampling for DPS can serve as an alternative to DPS sampling via standard pipetting and wet plasma in in vivo studies.
Keywords: Dried plasma spot (DPS) sampling; Plasma microsampling; Dried matrix card; LC-MS/MS; Ritonavir; Quantitative bioanalysis;

While capillary zone electrophoresis (CZE) has been used to produce very rapid and efficient separations, coupling these high-speed separations with mass spectrometry (MS) has been challenging. Now, with much faster and sensitive mass spectrometers, it is possible to take full advantage of the CZE speed and reconstruct the fast migrating peaks. Here are three high-speed CZE–MS analyses via an electrokinetically pumped sheath-flow interface. The first separation demonstrates CZE–ESI-MS of an amino acid mixture with a 2-min separation, >50,000 theoretical plates, low micromolar concentration detection limits, and subfemtomole mass detection limits (LTQ XL mass spectrometer). The second separation with our recently improved third-generation CE–MS interface illustrates a 20 amino acid separation in ∼7 min with an average over 200,000 plate counts, and results in almost-baseline resolution of structural isomers, leucine and isoleucine. The third separation is of a BSA digest with a reproducible CZE separation and mass spectrometry detection in 2 min. CZE–MS/MS analysis of the BSA digest identified 31 peptides, produced 52% sequence coverage, and generated a peak capacity of ∼40 across the 1-min separation window (Q-Exactive mass spectrometer).
Keywords: High-speed separation; Capillary zone electrophoresis; Electrokinetically pumped nanospray; Tandem mass spectrometry; Amino acids; Tryptic digest;

Pathogenic bacteria cause significant morbidity and mortality to humans. There is a pressing need to establish a simple and reliable method to detect them. Herein, we show that magnetic particles (MPs) can be functionalized by poly(diallyl dimethylammonium chloride) (PDDA), and the particles (PDDA-MPs) can be utilized as adsorbents for capture of pathogenic bacteria from aqueous solution based on electrostatic interaction. The as-prepared PDDA-MPs were characterized by Fourier-transform infrared spectroscopy, zeta potential, vibrating sample magnetometry, X-ray diffraction spectrometry, scanning electron microscopy, and transmission electron microscopy. The adsorption equilibrium time can be achieved in 3 min. According to the Langmuir adsorption isotherm, the maximum adsorption capacities for E. coli O157:H7 (Gram-negative bacteria) and L. monocytogenes (Gram-positive bacteria) were calculated to be 1.8 × 109 and 3.1 × 109  cfu mg−1, respectively. The bacteria in spiked mineral water (1000 mL) can be completely captured when applying 50 mg of PDDA-MPs and an adsorption time of 5 min. In addition, PDDA-MPs-based magnetic separation method in combination with polymerase chain reaction and capillary electrophoresis allows for rapid detection of 101  cfu mL−1 bacteria.
Keywords: Poly(diallyl dimethylammonium chloride); Magnetic particles; Polymerase chain reaction; Capillary electrophoresis; Detection; Pathogenic bacteria;

A method for the detection and confirmation of organic solvent extractable residues of the neutral, acidic, and basic analytes of the amphenicol class veterinary drugs and selected metabolites was developed and validated. Using a modified QuEChERS extraction with SPE cleanup and LC–MS/MS analysis, limits of detection and confirmation for the different analytes in bovine, equine, and porcine liver ranged from 0.1 ng/g for chloramphenicol to 1 ng/g for florfenicol amine. Tissue homogenization with an ammonium formate/EDTA solution and subsequent analyte partitioning against 7:3 acetonitrile:isopropanol solution and mixed-mode strong-cation exchange solid-phase extraction cartridge cleanup allowed for the extraction of all compounds from tissues with mean recoveries ranging from 50% (chloramphenicol 3-O-β-d-glucuronide) to 90% (thiamphenicol). Matrix effects ranged from greater than 85% suppression for florfenicol amine to 70% matrix enhancement for chloramphenicol 3-O-β-d-glucuronide. Quantitation and confirmation were accomplished using commercially available penta-deuterated chloramphenicol as internal standard and multiple reaction monitoring (MRM) of two or three transitions per target analyte. Method accuracy was greater than 15% for all compounds except the glucuronide metabolite. Intra-lab method repeatability estimates ranged from 73% RSD for chloramphenicol 3-O-β-d-glucuronide to 14% RSD for chloramphenicol. Only chloramphenicol 3-O-β-d-glucuronide and florfenicol amine at the low end of their calibration ranges (0.25 and 1 ng/g, respectively) did not meet AOAC recommended HorRatr guidelines for intra-lab repeatabilities. Preliminary tests show that the method's extraction protocol can be used to recover analytes of the β-agonists, corticosteroids, fluoroquinolones, sulfonamides, and tetracycline drug classes from the same matrices. Requirements for use in national chemical monitoring programs as a detection/confirmatory (florfenicol amine and chloramphenicol 3-O-β-d-glucuronide) and determinative/confirmatory (chloramphenicol, florfenicol, thiamphenicol) analytical methodology are met.
Keywords: Amphenicols; Chloramphenicol-glucuronide; Matrix effects; QuEChERS; Recovery;

One step affinity recovery of 3α-hydroxysteroid dehydrogenase from cloned Escherichia coli by Hailin Yang; Yanan Fang; Zhizhen Wang; Ling Zhang (79-84).
3α-Hydroxysteroid dehydrogenase (3α-HSD), from Comamonas Testosterone, catalyze reversibly the oxidoreduction of 3α-hydroxyl groups of the steroid hormones. The gene encoding 3α-HSD (hsdA) from Comamonas Testosterone was expressed in Escherchia coli BL21 (DE3). A protocol for recovering 3α-HSD based on affinity strategy was designed and employed. Deoxycholic acid was chosen as the affinity ligand, and it was linked to Sepharose 4B with the aid of the spacers as cyanuric chloride and ethanediamine. With this specific affinity medium, the enzyme recovery process consisted of only one chromatography step to capture 3α-HSD. The target protein, analyzed on HPLC Agilent SEC-5 column, was of 94% pure among the captured protein, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 8.8% of crude extracted proteins; the recovery yield of 3α-HSD was 73.2%. 3α-HSD was revealed as a non-covalent homodimer with molecular mass of ∼56 kDa by 15.0% SDS-PAGE analysis and SE-HPLC analysis. The desorption constant K d and the theoretical maximum absorption Q max on the affinity medium were 4.5 μg/g medium and 21.3 mg/g medium, respectively.
Keywords: 3α-Hydroxysteroid dehydrogenase; Affinity recovery; Purification; Ligand; E. coli;

A UHPLC–MS/MS bioanalytical assay for the determination of BMS-911543, a JAK2 inhibitor, in human plasma by Jane Liu; Long Yuan; Guowen Liu; Jim X. Shen; Anne-Françoise Aubry; Mark E. Arnold; Qin C. Ji (85-91).
Herein we report a rapid, accurate and robust UHPLC–MS/MS assay for the quantitation of BMS-911453, a Janus kinase 2 inhibitor under clinical development for the treatment of myeloproliferative disorders, in human plasma. A systematic method development approach was used to optimize the mass spectrometry, chromatography, and sample extraction conditions, and to minimize potential bioanalytical risks. The validated method utilizes stable-isotope labeled 13C4-BMS-911543 as the internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved within 2 min on a Zorbax Extend-C18 column with an isocratic elution. BMS-911543 and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The assay range was from 1 to 500 ng/mL, and the standard curve was fitted with 1/x 2 weighted linear regression. The intra-assay precision was within 5.0% CV and the inter-assay precision was within 2.6% CV. The inter-assay mean accuracy, expressed as percents of theoretical, was between 99.8% and 102.3%. The assay has high recovery (∼80%) and minimal matrix effect (0.95–1.00). BMS-911543 was stable in human plasma for at least 24 h at room temperature, 90 days at −20 °C, and following three freeze–thaw cycles. The validated method was successfully applied to sample analysis in clinical studies.
Keywords: Liquid–liquid extraction; LC–MS/MS; Quantitation; High-throughput; Automation; 96-Well format;

A liquid chromatography with tandem mass spectrometry method for simultaneous determination of UTL-5g and its metabolites in human plasma by Jiajiu Shaw; Richard Wiegand; Jianmei Wu; Xun Bao; Frederick Valeriote; Jing Li (92-98).
UTL-5g is a novel small-molecule TNF-α inhibitor under investigation as both a chemoprotective and radioprotective agent. Animal studies showed that pretreatment of UTL-5g protected kidney, liver, and platelets from cisplatin-induced toxicity. In addition, UTL-5g reduced liver and lung injuries induced by radiation in vivo. Although a number of preclinical studies have been conducted, a validated bioanalytical method for UTL-5g in human plasma has not been published. In this work, a sensitive and reproducible reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) assay was developed and validated for the determination of UTL-5g and its metabolites, 5-methylisoxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA), in human plasma. The method involves a simple methanol precipitation step followed by injection of the supernatant onto a Waters 2695 HPLC system coupled with a Waters Quattro Micro™ triple quadrupole mass spectrometer. Chromatographic separation was accomplished using a Waters Nova-Pak C18 column maintained at 30 °C, running at gradient mode with mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.2 mL/min. The analytes were monitored under positive electrospray ionization (ESI). Quantitation of these compounds in plasma was linear from 0.05 to 10 μM. The lower limit of quantitation (LLOQ) was 0.05, 0.1, and 0.2 μM for UTL-5g, ISOX and DCA, respectively. The accuracy and intra-and inter-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method provides a practical tool to measure and characterize the plasma concentration-time profiles for UTL-5g and its metabolites, ISOX and DCA.
Keywords: UTL-5g; Bioanalytical method validation; LC–MS/MS;

In vitro detection of small molecule metabolites excreted from cancer cells using a Tenax TA thin-film microextraction device by Takuma Nozoe; Shigemi Goda; Roman Selyanchyn; Tao Wang; Kohji Nakazawa; Takeshi Hirano; Hidetaka Matsui; Seung-Woo Lee (99-107).
We developed a new device for the in vitro extraction of small molecule metabolites excreted from cancer cells. The extraction device, which was biocompatible and incubated with cancer cells, consists of a thin Tenax TA film deposited on the surface of a cylindrical aluminum rod. The Tenax TA solid phase was utilized for the direct extraction and preconcentration of the small molecule metabolites from a cell culture sample. The device fabrication and the metabolite extraction were optimized, tested, and validated using HeLa cell cultures. Comparison of metabolic profiles with the control measurement from the culture medium enabled detection of metabolites that were consumed or produced by the cell culture. Tentative identification and semi-quantitative investigation of the excreted metabolites were performed by GC–MS analysis. The proposed approach can be a valuable tool for the characterization of low-volatile cancer cell metabolites that are not covered by use of conventional methods based on headspace solid phase microextraction.
Keywords: Molecular cancer biomarkers; Cell culture; Thin-film microextraction; GC–MS;

A simple, sensitive and reliable ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for simultaneous quantitation of seven coumarins, the bio-active ingredients of Huo Luo Xiao Ling Dan (HLXLD), in rat plasma. The liquid–liquid extraction method with ether–dichloromethane (2:1, v/v) was used to prepare the plasma samples. Analytes and internal standard (IS) of bifendate were separated on a Shim-pack XR-ODS column (75 mm × 3.0 mm, 2.2 μm particles) using gradient elution with the mobile phase consisting of methanol and 0.05% formic acid in water at a flow rate of 0.4 mL/min. Detection was performed on a triple quadrupole (TQ) tandem mass spectrometry equipped with an electrospray ionization source in the positive ionization and multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 0.03–0.25 ng/mL for all the analytes. Intra- and inter-day precision and accuracy of the seven analytes were well within acceptance criteria (15%). The matrix effect and the mean extraction recoveries of the analytes and IS from rat plasma were all within satisfaction. The validated method has been successfully applied to compare pharmacokinetic profiles of the seven active ingredients in rat plasma between normal and arthritic rats after oral administration of HLXLD, Angelica pubescens extract and Notopterygium incisum extract, respectively. Results showed that there were remarkable differences in pharmacokinetic properties of the analytes among the different groups.
Keywords: Arthritis; Huo Luo Xiao Ling Dan; Coumarins; UPLC–MS/MS; Pharmacokinetics;

The detection of 25-hydroxyvitamin D at low levels in biological samples is facilitated by the use of chemical derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in concert with liquid chromatography–tandem mass spectrometry (LC–MS/MS). This mode of analysis is notably hampered by chromatographic co-elution of 25-hydroxyvitamin D3 (25OHD3) and its C-3 epimer (C3epi). The objective of this work was to improve upon current LC–MS/MS methods used for the analysis of PTAD-derivatized 25-hydroxyvitamin D3 by resolving it from C3epi. Additionally, the applicability of this method in human serum and murine skin was investigated. C18 columns of increasing length and varying particle sizes were assessed for performance using a mixed standard of PTAD-derivatized 25OHD3 and C3epi. Serum samples were processed using solid phase extraction, and skin was powdered and extracted for lipophilic compounds. The samples were derivatized with PTAD and subsequently analyzed using isotope dilution LC–MS/MS with atmospheric pressure chemical ionization operated in positive mode. Near baseline resolution of PTAD-25OHD3 from PTAD-C3epi was achieved on a 250 mm C18 column with 3 μm sized particles. This separation allowed for detection and quantification of both metabolites in serum and skin samples. PTAD-C3epi represented a significant confounding analyte in all samples, and comprised up to 20% of the status measurement in skin. This method is a significant improvement on the chromatography of PTAD-derivatized vitamin D metabolites that could greatly influence the assessment of vitamin D status and C3epi biology in low abundance samples.
Keywords: 25-Hydroxyvitamin D analysis; C3-epi-25-hydroxyvitamin D; HPLC–MS/MS; PTAD; Vitamin D;