Separation and identification of monoglucuronides of vitamin D3 metabolites in urine by derivatization-assisted LC/ESI-MS/MS using a new Cookson-type reagent.

A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method was developed using a new Cookson-type reagent, 4-[4-(1-pipelidinyl)phenyl]-1,2,4-triazoline-3,5-dione (PIPTAD), to analyze the monoglucuronides (Gs) of vitamin D3 metabolites in human urine. The G of 23S,25-dihydroxyvitamin D3 [23,25(OH)2D3] was previously found as a major metabolite of vitamin D3 in the urine, but its conjugation position remained undetermined. Determination of the position was an important research issue to clarify the whole picture of the excretion of surplus 25-hydroxyvitamin D3 [25(OH)D3, the circulating form of vitamin D3] in humans. After the pretreated urine sample was derivatized with PIPTAD, the peak corresponding to the G of 23,25(OH)2D3 was satisfactorily separated from the urine-derived interfering substances on reversed-phase LC, which could not be achieved by using the previous analogous reagent, DAPTAD. The PIPTAD-derivatized Gs of the vitamin D3 metabolites provided characteristic product ions useful for identifying the conjugation positions during the MS/MS. Accordingly, we successfully determined the glucuronidated position of 23,25(OH)2D3 to be the C23-hydroxy group. The developed method also enabled the simultaneous detection of Gs of 25(OH)D3 and 24R,25-dihydroxyvitamin D3 as well as 23,25(OH)2D3-23-G without interference from the urine components.

Evaluation of thumbnail clipping as a specimen for retrospectively assessing average production of testosterone.

BACKGROUND: The chronic abnormal production of testosterone (T) is associated with many disorders in men. Fingernail clippings might be more suited for the diagnosis and medium-to-long term therapeutic monitoring for the T-related chronic disorders than the blood-derived specimens. The objective of this study was to characterize a thumbnail clipping as the specimen for assessing the several months-old T status. METHODS: Thumbnail clippings from various subjects were analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry to evaluate the gender difference, and changes caused by aging and androgen deprivation therapy (ADT) in the thumbnail T concentration. RESULTS: There was an evident gender difference in the thumbnail T concentrations [male; 2.55 ± 0.85 ng/g and female; 0.48 ± 0.29 ng/g, mean ± SD (n = 25 each), Welch t-test]. The thumbnail T concentrations significantly decreased with age in men (n = 268, Scheffé F-test), which was similar to those of the free or bioavailable T in serum/plasma. The thumbnail T concentrations sharply decreased by a 6-months ADT (especially the effect of the luteinizing hormone-releasing hormone agonist/antagonist) for patients with prostate cancer (n = 10). CONCLUSIONS: The thumbnail clipping can be a specimen to retrospectively assess the average T production.

An LC/MS/MS method for quantifying testosterone and dehydroepiandrosterone sulfate in four different serum samples during a single run.

Simultaneous measurements of the circulating testosterone (TS) and dehydroepiandrosterone sulfate (DHEAS) are deemed to be helpful for the assessment of men's health. Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) is the most reliable methodology for this purpose; however, it has room for improvement in analysis throughput. In this study, a quadruplicate of the Girard reagents was used to develop an LC/ESI-MS/MS method capable of quantifying TS and DHEAS in four different serum samples in a single run. The four serum samples were separately pretreated, derivatized with one of four Girard reagents, and then combined. The LC/ESI-MS/MS analysis of the combined sample provided the androgen concentrations of four serum samples in parallel. The method had practical measuring ranges, in which good precision and accuracy, as well as negligible matrix effects were verified. The speed-up capability of the developed method was evaluated through the analysis of ten batches of serum samples (total 40 samples); the method saved a 60% post-pretreatment analysis time compared to the non-derivatization method for 40 samples.

A method for determination of aldosterone concentrations of six adrenal venous serum samples during a single LC/ESI-MS/MS run using a sextet of Girard reagents.

The accurate quantification of aldosterone (ALD) in adrenal tributary venous serum/plasma collected by a super-selective adrenal venous sampling (ssAVS) technique has become a definitive procedure to identify the forms (laterality) of primary aldosteronism (PA) and the location of the affected segment(s), and to subsequently make the treatment decision. In this study, a stable isotope-dilution LC/ESI-MS/MS-based method was developed and validated for the determination of the ALD concentrations of the six ssAVS serum samples during a single run. A sextet of Girard reagents was used to convert ALD to the derivatives having different masses for each sample. Based on the validation tests, satisfactory specificity, precision and accuracy were observed and the matrix effect was found to be acceptable. The developed method was used for the analysis of the ssAVS serum samples from eight PA patients. The batch analysis of the six samples permitted the direct comparison of the ALD concentrations between the samples and enhanced to determine the laterality and location of the affected segment(s). The total analysis time after the sample purification was also reduced to 1/3 for the 24 samples by the sample-sextuplex procedure, which was also an advantage of the developed method.

Derivatization-based quadruplex LC/ESI-MS/MS method for high throughput quantification of serum dehydroepiandrosterone sulfate.

The quantification of the circulating dehydroepiandrosterone sulfate (DHEAS) might be of diagnostic help for several diseases. For the DHEAS quantification, LC/ESI-MS/MS has the advantage of a high specificity compared with immunoassay, whereas LC/ESI-MS/MS has room to improve the analysis throughput. One of the promising solutions to enhance the analysis throughput is sample-multiplexing in the same injection, which can reduce the total LC/ESI-MS/MS run time. In this study, a quadruplex LC/ESI-MS/MS method was developed to quantify DHEAS in four different serum samples in a single run. After the four samples were separately deproteinized and derivatized with one of four Girard reagents (Girard reagent T, P and their isotopologs), the resulting samples were mixed, then injected into the LC/ESI-MS/MS. The applicability and advantage of the developed method were evaluated based on the analysis of nine batches of serum samples from healthy subjects (total 36 samples). The limit of quantitation was 0.050 µg/ml, which was sensitive enough for clinical laboratory use. The method was precise (intra- and inter-assay RSDs ≤ 3.6%), accurate (94.4-108.1%) and robust for the matrix effects. The analysis time was also shortened by about 60% for 36 samples by the introduced method compared with the conventional method.

Derivatization-based sample-multiplexing for enhancing throughput in liquid chromatography/tandem mass spectrometry quantification of metabolites: an overview.

The quantification of metabolites in various samples, including body fluids, tissues, cells, and foodstuffs, contributes to our understanding of their biological activities and roles in the body, diagnosis for many diseases, drug and biomarker discovery, and many aspects of human health. Liquid chromatography (LC)/tandem mass spectrometry (MS/MS) is the most powerful and reliable methodology for the quantification of metabolites due to its high specificity and sensitivity, and broad coverage of various compounds. Derivatization often makes the quantification power of LC/MS/MS stronger due to the desirable LC behavior and enhanced MS/MS detectability of the derivatized metabolites. On the other hand, LC/MS/MS-based quantification has room for improvement regarding its analysis throughput. Derivatization is also a promising approach to overcome this drawback; the multiplexing of samples in the same LC/MS/MS injection, which is achieved by derivatization of multiple samples with multiple well-designed reagents, can enhance the throughput. Based on this background, this article reviews the derivatization-based sample-multiplexing strategy, especially the characteristics and applications of the derivatization reagents, for the LC/MS/MS quantification of metabolites. This strategy has been used for the relative and absolute quantification of a variety of metabolites, and expansion of the coverage of metabolites.

3-Epi-25-hydroxyvitamin D3 is a poor substrate for SULT2A1: Analysis of its 3-sulfate in cord plasma and recombinant human SULT2A1 incubate.

A variety of metabolites derived from 25-hydroxyvitamin D3 [25(OH)D3], including its 3-epimer [Epi-25(OH)D3] and 3-O-sulfate [25(OH)D3-3S], is found in human plasma/serum. We hypothesized that the 3-O-sulfate of Epi-25(OH)D3 [Epi-25(OH)D3-3S] might be present in plasma/serum. Clarifying this point could improve our understanding of the metabolism of vitamin D3. In this study, we first carefully analyzed the cord plasma samples by derivatization-assisted liquid chromatography/electrospray ionization-tandem mass spectrometry and demonstrated the occurrence of Epi-25(OH)D3-3S in the plasma. However, the concentration ratio of Epi-25(OH)D3-3S to 25(OH)D3-3S (sulfated form) was infinitely lower than the ratio of Epi-25(OH)D3 to 25(OH)D3 (unconjugated form). To determine what caused this result, we next performed an in vitro experiment of the 3-O-sulfation for 25(OH)D3 and Epi-25(OH)D3 using the recombinant human sulfotransferase (SULT) 2A1. This in vitro experiment revealed that Epi-25(OH)D3 is a poor substrate for the 3-O-sulfation catalyzed by SULT2A1 as compared to 25(OH)D3. This substrate specificity of SULT2A1 would be the main cause for the result obtained from the analysis of the cord plasma samples.

Enhancing LC/ESI-MS/MS Throughput for Plasma Bile Acid Assay by Derivatization-based Sample-Multiplexing.

Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) enables the accurate and precise quantification of various analytes at very low concentrations, but it has room for improvement in analysis throughput. Multiplexing of samples in the same injection could be a promising procedure for enhancing the analysis throughput. This could be achieved by derivatization of the multiple samples with multiple isotopologous reagents. In this study, a sample-multiplexed LC/ESI-MS/MS assay using the 1-[(4-dimethylaminophenyl)carbonyl]piperazine (DAPPZ) isotopologues (2H0-, 2H3-, and 2H6-forms) was developed and validated for the simultaneous determination of primary bile acids in three different plasma samples in a single run. The developed method had satisfactory intra- and inter-assay precisions (≤ 2.3 and ≤ 4.2%, respectively) and accuracy (99.0 - 100.3%), and could reduce the total LC/ESI-MS/MS run time by more than 60% for 42 samples compared to the conventional method. Thus, the derivatization with the DAPPZ isotopologues worked well for enhancing the throughput of the LC/ESI-MS/MS assay of the bile acids.

Sample-multiplexing by derivatization using multiple analogous reagents for enhancing throughput in LC/ESI-MS/MS assay of steroids: Plasma 17α-hydroxyprogesterone as an example.

The measurements of steroids in biological fluids are of importance for the diagnosis and treatment of many diseases. Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has a high specificity and accuracy for the steroid analysis, whereas it has a lower analysis throughput, which could become a big issue in clinical practice. One of the promising solutions to this issue is the multiplexing of samples in the same injection. In this study, the utility of the sample-multiplexing by the derivatization using multiple analogous reagents was evaluated for enhancing the throughput of the LC/ESI-MS/MS assays of steroids. The plasma 17α-hydroxyprogesterone (17OHP), which is a diagnostic marker for the 21-hydroxylase deficiency, was chosen as the model analyte. The four plasma samples (20 µL each) were separately derivatized with one of four different analogous Girard-type reagents, combined, then injected together into the LC/ESI-MS/MS. By this method, four plasma samples could be analyzed within a single LC run. The developed method could significantly reduce the total LC run time (about 2/5 for 32 samples, compared with the conventional method) with a satisfactory sensitivity (lower limit of quantification 0.5 ng/mL), precision (intra- and inter-assay RSDs ≤ 4.0% and ≤ 3.5%, respectively) and accuracy (97.6-106.7%), and negligible matrix effect. The developed method had a satisfactory applicability for the quantification of 17OHP in the cord plasma samples.

Quantitative MALDI-MS/MS assay for serum cortisol through charged derivatization.

Cortisol (CRT), the main glucocorticoid in humans, plays a crucial role in many physiological processes, therefore, the measurement of its serum level is of great clinical significance. Matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS) might be an effective approach for the quantification of CRT in serum due to its attractive properties, i.e., high specificity, ease of use, ruggedness and rapid analysis. In this study, we developed a method to quantify the serum CRT by MALDI-MS/MS. This method employed the derivatization using a Girard-type reagent, 1-(hydrazinocarbonylmethyl)isoquinolinium chloride, which compensated for the lack of sensitivity. This method enabled the reproducible quantification of the serum CRT using a 20-µL sample (intra- and inter-assay relative standard deviations, 7.4% or lower), and the measurable range was 25-500 ng/mL. The serum CRT concentrations determined by the newly-developed MALDI-MS/MS method agreed well with those by liquid chromatography/electrospray ionization-MS/MS or electrochemiluminescence immunoassay. The MALDI-MS/MS method was used for the analysis of peripheral venous serum samples of healthy subjects and adrenal venous serum samples of patients with primary aldosteronism, and satisfactory results were obtained.

Identification of conjugation positions of urinary glucuronidated vitamin D3 metabolites by LC/ESI-MS/MS after conversion to MS/MS-fragmentable derivatives.

A liquid chromatography/electrospray ionization-tandem mass spectrometry-based method was developed for the identification of the conjugation positions of the monoglucuronides of 25-hydroxyvitamin D3 [25(OH)D3 ] and 24,25-dihydroxyvitamin D3 [24,25(OH)2 D3 ] in human urine. The method employed derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione to convert the glucuronides into fragmentable derivatives, which provided useful product ions for identifying the conjugation positions during the MS/MS. The derivatization also enhanced the assay sensitivity and specificity for urine sample analysis. The positional isomeric monoglucuronides, 25(OH)D3 -3- and -25-glucuronides, or 24,25(OH)2 D3 -3-, -24- and -25-glucuronides, were completely separated from each other under the optimized LC conditions. Using this method, the conjugation positions were successfully determined to be the C3 and C24 positions for the glucuronidated 25(OH)D3 and 24,25(OH)2 D3 , respectively. The 3-glucuronide was not present for 24,25(OH)2 D3 , unlike 25(OH)D3 , thus we found that selective glucuronidation occurs at the C24-hydroxy group for 24,25(OH)2 D3 .

Changes in Polyamine Content in Rice Bran due to Fermentation with Aspergillus oryzae Analyzed by LC/ESI-MS/MS Combined with Derivatization.

Many studies have demonstrated that the dietary supplementation of polyamines, especially spermidine (SPD), prevents age-related diseases. Rice bran is rich in polyamines and their amounts could be increased by fermentation with Aspergillus oryzae (A. oryzae). In this study, we developed a method for the determination of putrescine (PUT), SPD and spermine (SPM) in rice bran samples by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) after derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). The derivatization improved the LC retention and ESI-MS/MS detectability of the polyamines, and consequently enabled precise and accurate quantification. Using this method, we found that the SPD content increased to 158% due to fermentation with A. oryzae, while the content of PUT and SPM decreased. SPD is known as the polyamine playing a central role in cell proliferation and growth, and therefore has health benefits. The fermented rice bran might be a good material for functional foods aimed at SPD supplementation.

A Method for Quantification of Tetrahydroglucocorticoid Glucuronides in Human Urine by LC/MS/MS with Isotope-coded Derivatization.

The determination of urinary tetrahydroglucocorticoid (THGC) glucuronides might prove helpful in the diagnosis, pathophysiological analysis and assessment of the therapeutic efficacy of the diseases caused by abnormal cortisol secretion. We developed and validated a method for the determination of the THGC glucuronides in human urine using liquid chromatography/electrospray ionization (ESI)-tandem mass spectrometry not requiring enzymatic hydrolysis. The method employed a derivatization using an ESI-enhancing reagent for carboxylic acids, 1-[(4-dimethylaminophenyl)carbonyl]piperazine (DAPPZ), and its isotopologue, 2H4-DAPPZ. The deproteinized urine samples were derivatized with DAPPZ. The 2H4-DAPPZ-derivatized standards of known amounts were then added to the DAPPZ-derivatized urine samples and served as the internal standards. The DAPPZ-derivatization enhanced the assay sensitivity and reduced the sample volume, and the use of 2H4-DAPPZ significantly improved the assay accuracy. The developed method enabled the separate quantification and profiling of the urinary THGC glucuronides and had a satisfactory application for the real sample analysis.

A method for determination of aldosterone in adrenal tributary venous serum by derivatization using Girard P reagent isotopologues followed by LC/ESI-MS/MS.

The quantification of aldosterone (ALD) in adrenal tributary venous blood serum/plasma combined with the super-selective adrenal venous sampling (ssAVS) technique is recognized as a definitive procedure for differentiation of the forms of primary aldosteronism (PA), identification of the affected segment(s) and operating decision-making. In this study, an enhanced throughput and sensitive method was developed and validated for the quantification of ALD in ssAVS serum samples by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using the Girard P reagent (GP) isotopologues (2H0- and 2H5-GP). The right and left adrenal serum samples were separately pretreated and derivatized with either isotopologue. The two samples were then combined and injected together for LC/ESI-MS/MS analysis. Based on the mass differences between the isotopologues, the derivatized ALD in the two samples were quantified within a single run. This method enabled the reproducible (intra- and inter-assay relative standard deviations, 6.6% or lower) and accurate (98.2-107.0%) quantification of the serum ALD using a 25-µL sample, and the lower limit of quantification was 1.0 ng/mL. The developed method was used for the analysis of 11 pairs of ssAVS serum samples (total of 22 samples) of patients with ALD-producing adenoma and proven to have a satisfactory applicability; this method enabled the identification of the affected adrenal and the determination of the laterality of PA, and reduced the analysis time to about 2/3 compared to the previous method for 22 samples.

Chemical Synthesis of Rare Natural Bile Acids: 11α-Hydroxy Derivatives of Lithocholic and Chenodeoxycholic Acids.

A method for the preparation of 11α-hydroxy derivatives of lithocholic and chenodeoxycholic acids, recently discovered to be natural bile acids, is described. The principal reactions involved were (1) elimination of the 12α-mesyloxy group of the methyl esters of 3α-acetate-12α-mesylate and 3α,7α-diacetate-12α-mesylate derivatives of deoxycholic acid and cholic acid with potassium acetate/hexamethylphosphoramide; (2) simultaneous reduction/hydrolysis of the resulting △11 -3α-acetoxy and △11 -3α,7α-diacetoxy methyl esters with lithium aluminum hydride; (3) stereoselective 11α-hydroxylation of the △11 -3α,24-diol and △11 -3α,7α,24-triol intermediates with B2 H6 /tetrahydrofuran (THF); and (4) selective oxidation at C-24 of the resulting 3α,11α,24-triol and 3α,7α,11α,24-tetrol to the corresponding C-24 carboxylic acids with NaClO2 catalyzed by 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and NaClO. In summary, 3α,11α-dihydroxy-5ß-cholan-24-oic acid and 3α,7α,11α-trihydroxy-5ß-cholan-24-oic acid have been synthesized and their nuclear magnetic resonance (NMR) spectra characterized. These compounds are now available as reference standards to be used in biliary bile acid analysis.

Improved sensitivity of serum/plasma 1α,25-dihydroxyvitamin D quantification by DAPTAD derivatization.

BACKGROUND: Although immunoassays have several limitations such as the cross-reactivities of antibodies, such techniques are widely used for serum/plasma 1,25(OH)2D quantification. An accurate method is required for the determination of the 1,25(OH)2D status. METHODS: We designed a serum/plasma 1,25(OH)2D quantification method using LC-MS/MS. Immunoaffinity extraction (IE) and the recently developed Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) were used for sample preparation and derivatization, respectively. Analytical and pre-analytical validations were performed. Serum 1,25(OH)2D3 concentrations were determined in 232 healthy Japanese individuals. RESULTS: The intra- and inter-assay CVs for 1,25(OH)2D3 were 5.2% and 7.0%, respectively. The limit of quantification for 1,25(OH)2D3 was 7.1pg/ml. Rheumatoid factor (RF) at concentrations below 517IU/ml did not affect serum 1,25(OH)2D analysis. No significant differences were observed for various blood collection tubes, repeated freeze-thaw cycles, whole blood standing time, or serum storage time. A strong correlation between LC-MS/MS and radioimmunoassay (RIA) was observed (r=0.786), but serum 1,25(OH)2D concentrations obtained from RIA were 2-fold higher than those obtained from LC-MS/MS. Serum 1,25(OH)2D3 concentrations by LC-MS/MS were 18.7-53.9pg/ml. CONCLUSION: A highly sensitive and selective LC-MS/MS-based serum/plasma 1,25(OH)2D quantification method was developed using IE and DAPTAD derivatization. This method will enable the accurate determination of serum/plasma 1,25(OH)2D concentrations in the clinical setting.

Unconjugated bile acids in rat brain: Analytical method based on LC/ESI-MS/MS with chemical derivatization and estimation of their origin by comparison to serum levels.

Although some studies have revealed the implication of bile acids (BAs) and neurological diseases, the levels and origin of the BAs in the brain are not fully understood. In this study, we first developed and validated a sensitive and specific method for the determination of three unconjugated BAs [cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)] in the rat brain by liquid chromatography/electrospray ionization-tandem mass spectrometry combined with chemical derivatization. The measured brain concentrations (mean±standard deviation, n=10) of normal rats were 58.7±48.8, 14.2±11.7 and 13.2±8.7ng/g tissue for CA, CDCA and DCA, respectively. For their origin, we developed the hypothesis that they might be mostly derived from the periphery. To test this hypothesis, the brain BA levels were compared with the serum levels. The brain levels had high correlations with the serum levels, and were always lower than the serum levels for the three unconjugated BAs. Furthermore, the higher brain-to-serum concentration ratios were found for the BAs with higher logD values (higher lipophilicity). Moreover, the brains of the rats intraperitoneally administered with deuterium-labeled CA and CDCA were also analyzed; the deuterium-labeled BAs were detected in the brain of the rats administered with these compounds. Based on all the results, we concluded that the BAs found in the brain are mostly derived from the periphery and the major mechanism for the transportation of the unconjugated BAs to the brain is by passive diffusion.

Isotope-coded derivatization based LC/ESI-MS/MS methods using a pair of novel reagents for quantification of hydroxycinnamic acids and hydroxybenzoic acids in fermented brown rice product.

Hydroxycinnamic acids (HCAs) and hydroxybenzoic acids (HBAs) are antioxidant phytochemicals found in rice and effective for the prevention of human diseases including cancer. FBRA, which is a functional food manufactured by fermenting brown rice and rice bran with Aspergillus oryzae, has been demonstrated to have chemopreventive effects against carcinogenesis in various organs. In this study, we developed methods for the relative and absolute quantification of ferulic acid, sinapic acid, caffeic acid, protocatechuic acid and syringic acid in the FBRA and raw material (RM; unfermented brown rice and rice bran) samples by LC/ESI-MS/MS combined with derivatization using a newly developed reagent, N-(2-aminoethyl)-4-(diethylamino)benzamide (ADB) and its deuterium-coded analog, d-ADB. For the relative quantification, the FBRA and RM samples were derivatized with ADB and d-ADB, respectively, then the resulting derivatives were mixed and subjected to LC/ESI-MS/MS; by this method, we found that the fermentation process significantly increased the free HCA and HBA contents. The HCA and HBA contents in the FBRA were also determined, in which the d-ADB-derivatized standards of known amounts were used as the internal standards. The ADB-derivatization enabled the sensitive and specific detection, and the use of d-ADB significantly improved the assay precision.

Enhancing analysis throughput, sensitivity and specificity in LC/ESI-MS/MS assay of plasma 25-hydroxyvitamin D3 by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) isotopologues.

The plasma/serum concentration of 25-hydroxyvitamin D3 [25(OH)D3] is a diagnostic index for vitamin D deficiency/insufficiency, which is associated with a wide range of diseases, such as rickets, cancer and diabetes. We have reported that the derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) works well in the liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) assay of the serum/plasma 25(OH)D3 for enhancing the sensitivity and the separation from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3]. However, enhancing the analysis throughput remains an issue in the LC/ESI-MS/MS assay of 25(OH)D3. The most obvious restriction of the LC/MS/MS throughput is the chromatographic run time. In this study, we developed an enhanced throughput method for the determination of the plasma 25(OH)D3 by LC/ESI-MS/MS combined with the derivatization using the triplex (2H0-, 2H3- and 2H6-) DAPTAD isotopologues. After separate derivatization with 1 of 3 different isotopologues, the 3 samples were combined and injected together into LC/ESI-MS/MS. Based on the mass differences between the isotopologues, the derivatized 25(OH)D3 in the 3 different samples were quantified within a single run. The developed method tripled the hourly analysis throughput without sacrificing assay performance, i.e., ease of pretreatment of plasma sample (only deproteinization), limit of quantification (1.0ng/mL when a 5µL-plasma was used), precision (intra-assay RSD≤5.9% and inter-assay RSD≤5.5%), accuracy (98.7-102.2%), matrix effects, and capability of separating from an interfering metabolite, 3-epi-25(OH)D3. The multiplexing of samples by the isotopologue derivatization was applied to the analysis of plasma samples of healthy subjects and the developed method was proven to have a satisfactory applicability.