Comparative evaluation of new Cookson‐type reagents for LC/ESI‐MS/MS assay of 25‐hydroxyvitamin D3 in neonatal blood samples

The screening of vitamin D deficiency in neonatal infants, which is based on the blood 25‐hydroxyvitamin D₃ [25(OH)D₃] quantification, is important for the early detection, diagnosis and health risk assessment of several diseases. In this study, two new Cookson‐type reagents, 4‐(4‐diethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DEAPTAD) and 4‐(6‐quinolyl)‐1,2,4‐triazoline‐3,5‐dione, were designed and synthesized, then compared with the previous reagents, 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) and 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DAPTAD), in terms of sensitivity and specificity in the assay of 25(OH)D₃ in neonatal blood samples by liquid chromatography/electrospray ionization–tandem mass spectrometry. Among the reagents, DEAPTAD was found to be the most promising. The limit of detection (0.38 fmol on the column) of the DEAPTAD‐derivatized 25(OH)D₃ was 60 and 2 times lower than those of the intact 25(OH)D₃ and the PTAD derivative, respectively. 25(OH)D₃ was more clearly detected in the plasma sample as the DEAPTAD derivative than the DAPTAD derivative owing to the lower background noise. DEAPTAD derivatization was also useful for the separation of 25(OH)D₃ from a potent interfering metabolite, 3‐epi‐25‐hydroxyvitamin D₃. By using DEAPTAD, a trace amount of 25(OH)D₃ in dried blood spots was reproducibly determined without interference from coexisting compounds. Thus, DEAPTAD was proved useful in the measurement of 25(OH)D₃ in neonatal blood samples. Copyright © 2015 John Wiley & Sons, Ltd.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D₃ [25(OH)D₃, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D₃ in ESI‐MS/MS and to separate 25(OH)D₃ from 3‐epi‐25‐hydroxyvitamin D₃ [3‐epi‐25(OH)D₃], a potent interfering metabolite. 25(OH)D₃ was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB^® cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D₄ was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D₃ in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D₃.     

Development and application of a UHPLC‐MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17‐OH‐pregnenolone, progesterone, 17‐OH‐progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11‐deoxycortisol, 11‐deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra‐high‐performance liquid chromatography–tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra‐high‐performance liquid chromatography–tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922–0.9986, and the limits of detection and limits of quantification were in the range of 0.002–2 and 0.0055–5.5 ng ml⁻¹, respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml⁻¹) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml⁻¹ free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25–51.26 ng ml⁻¹) and cortisol (range 32.57–52.24 ng ml⁻¹), followed by 17‐OH‐pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a highly sensitive LC‐MS/MS method for simultaneous quantitation of nortriptyline and 10‐hydroxynortriptyline in human plasma: application to a human pharmacokinetic study

A highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10‐hydroxynortriptyline (OH‐NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract NTP, OH‐NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH‐NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C₁₈ column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH‐NTP. A linear response function was established for the range of concentrations 1.09–30.0 ng/mL (r > 0.998) for both NTP and OH‐NTP. The intra‐ and inter‐day precision values for NTP and OH‐NTP met the acceptance as per FDA guidelines. NTP and OH‐NTP were stable in a battery of stability studies, i.e. bench‐top, auto‐sampler and freeze–thaw cycles. The developed assay was applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease by HPLC–MS/MS method

Fat‐soluble vitamins play a pivotal role in the progression of atherosclerosis and the development of cardiovascular disease. Therefore, plasma monitoring of their concentrations may be useful in the diagnosis of these disorders as well as in the process of treatment. The study aimed to develop and validate an HPLC–MS/MS method for determination of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease. The analytes were separated on an HPLC Kinetex F5 column via gradient elution with water and methanol, both containing 0.1% (v/v) formic acid. Detection of the analytes was performed on a triple‐quadrupole MS with multiple reaction monitoring via electrospray ionization. The analytes were isolated from plasma samples with liquid–liquid extraction using hexane. Linearity of the analyte calibration curves was confirmed in the ranges 0.02–2 μg/mL for retinol, 0.5–20 μg/mL for α‐tocopherol, 5–100 ng/mL for 25‐hydroxyvitamin D2 and 2–100 ng/mL for 25‐hydroxyvitamin D3. Intra‐ and inter‐assay precision and accuracy of the method were satisfactory. Short‐ and long‐term stabilities of the analytes were determined. The HPLC‐MS/MS method was applied for the determination of the above fat‐soluble vitamin concentrations in patient plasma as potential markers of the cardiovascular disease progression.     

LC–MS/MS analysis of metformin,saxagliptin and 5‐hydroxy saxagliptin in human plasma and its pharmacokinetic study with a fixed‐dose formulation in healthy Indian subjects

A specific and rapid liquid chromatography–tandem mass spectrometry method is proposed for the simultaneous determination of metformin (MET), saxagliptin (SAXA) and its active metabolite, 5‐hydroxy saxagliptin (5‐OH SAXA) in human plasma. Sample preparation was accomplished from 50 μL plasma sample by solid‐phase extraction using sodium dodecyl sulfate as an ion‐pair reagent. Reversed‐phase chromatographic resolution of analytes was possible within 3.5 min on ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile and10.0 mm ammonium formate buffer, pH 5.0 (80:20, v /v) as the mobile phase. Triple quadrupole mass spectrometric detection was performed using electrospray ionization in the positive ionization mode. The calibration curves showed good linearity (r ² ≥ 0.9992) over the established concentration range with limit of quantification of 1.50, 0.10 and 0.20 ng/mL for MET, SAXA and 5‐OH SAXA respectively. The extraction recoveries obtained from spiked plasma samples were highly consistent for MET (75.12–77.84%), SAXA (85.90–87.84%) and 5‐OH SAXA (80.32–82.69%) across quality controls. The validated method was successfully applied to a bioequivalence study with a fixed‐dose formulation consisting of 5 mg SAXA and 500 mg MET in 18 healthy subjects. The reproducibility of the assay was demonstrated by reanalysis of 87 incurred samples.     

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)₂D₃] 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)₂D₃‐3‐, ‐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)₂D₃, respectively. The 3‐glucuronide was not present for 24,25(OH)₂D₃, unlike 25(OH)D₃, thus we found that selective glucuronidation occurs at the C24‐hydroxy group for 24,25(OH)₂D₃.     

Development,validation and comparison of four methods for quantifying endogenous 25OH‐D3 in human plasma

To meet the increasing clinical needs for 25‐hydroxyvitamin D3 (25OH‐D3) detection, the development of an efficient and accurate high‐performance liquid chromatography–mass spectrometry (HPLC–MS) method for plasma 25OH‐D3 quantitation is important. Since 25OH‐D3 is an endogenous compound, the lack of a plasma blank increases the difficulty of accurately quantifying 25OH‐D3. Selection of a method suitable for clinical monitoring among various methods for endogenous compound quantification is necessary. Methyl tert butyl ether was chosen for the sample treatment in a liquid–liquid extraction protocol. Water as a blank matrix, 5% human serum albumin in water as a blank matrix, surrogate analyte and background subtraction were designed to address the problem of a deficiency of a plasma blank. Four liquid chromatography–tandem mass spectrometry methods were fully validated to verify the advantages and limitations owing to regulatory deficiencies for endogenous compound validation. All four methods met the criteria and could be used to monitor clinical samples. Overall 30 human plasma samples were quantified in parallel using the four methods. The difference between any two methods was <12.6% and the total relative standard deviation was <5.2%. Background subtraction and 5% human serum albumin in water as a blank matrix may be better choices considering data quality, matrix similarity, cost and practicality.     

Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

Ricin, a plant‐derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks. Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl‐agarose (LA) beads. (b) Tryptic digestion. (c) LC‐MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences. Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM‐based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP‐II toxins, by applying multiplex format.     

Sensitive LC‐MS‐MS method for the determination of tiopronin in human plasma

A highly selective and sensitive LC‐MS‐MS method was developed and validated to quantify tiopronin in human plasma, using fudosteine as the internal standard (IS). L ‐Cysteine and 1,4‐dithiothreitol (DTT) were used as the reducer and the stabilizer to release and stabilify tiopronin from a dimmer and mix forms with endogenous thiols in the treatment of plasma samples. After a simple liquid–liquid extraction with ethyl acetate in acidic condition, the post‐treatment samples were analyzed on a C₁₈ column interfaced with a triple‐quadruple tandem mass spectrometer using negative electrospray ionization. Methanol and water (40:60, v/v) were used as the isocratic mobile phase, with 0.2% formic acid and 1.0 mM tris (hydroxymethyl) aminomethane (Tris) in water. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy and precision of measurements. The assay was linear over the concentration range 0.078–10 μg/mL. The correlation coefficients for the calibration curves ranged from 0.9980 to 0.9990. The intra‐ and inter‐day precisions, calculated from quality control samples, were not more than 10.49%. The method was employed in a pharmacokinetic study after oral administration of 200 mg tiopronin tablets to 24 healthy volunteers. Copyright © 2009 John Wiley & Sons, Ltd     

Two‐dimensional LC‐MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver

A two‐dimensional (2D) hydrophilic interaction liquid chromatography (HILIC) and reverse‐phase (RP) liquid chromatography (LC) system coupled with triple‐quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphatidylcholine in extracted biological samples. Briefly, the 2D HILIC‐RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C₁₈ column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however, the absolute matrix effect factor was reduced in 2D LC‐MS/MS system. Limits of detection of 2D LC‐MS/MS system were 2‐ to 3‐fold lower compared with one‐dimensional RPLC‐MS/MS. The recovery from the sample ranged from 84.5 to 110%. To summarize, the developed method was proven to be accurate and producible, as relative standard deviations remained <20% at three spiked levels. The efficiency of this newly developed system was applied to measure changes of lipids in the liver of mice after naphthalene treatment. Orthogonal projection to latent structures‐discriminant analysis discriminated the lipids from control and the treatment group. We concluded that 2D LC‐MS/MS is a promising method to assist lipidomic studies of complex biological samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of asperosaponin VI in rat plasma by HPLC‐ESI‐MS and its application to preliminary pharmacokinetic studies

Asperosaponin VI (also named akebia saponin D) is a typical bioactive triterpenoid saponin isolated from the rhizome of Dipsacus asper Wall (Dipsacaceae). In this work, a sensitive high‐performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC‐ESI‐MS) assay has been established for determination of asperosaponin VI in rat plasma. With losartan as the internal standard (IS), plasma samples were prepared by protein precipitation with methanol. Chromatographic separation was performed on a C₁₈ column with a mobile phase of 10 mm ammonium acetate buffer containing 0.05% formic acid–methanol (32 : 68, v/v). The analysis was performed on an ESI in the selected ion monitoring mode using target ions at m/z 951.4 for asperosaponin VI and m/z 423.2 for the IS. The calibration curve was linear over the range 3–1000 ng/mL and the lower limit of quantification was 3.0 ng/mL. The intra‐ and inter‐assay variability values were less than 9.5 and 7.8%, respectively. The accuracies determined at the concentrations of 3.0, 100.0, 300.0 and 1000 ng/mL for asperosaponin VI were within ±15.0%. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of asperosaponin VI. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of cefoperazone and sulbactam in serum by HPLC‐MS/MS: An adapted method for therapeutic drug monitoring in children

A rapid, accurate and specific high‐performance liquid chromatography–tandem mass spectrometry method has been validated for the simultaneous determination of cefoperazone and sulbactam in a small volume sample for children. A Shim‐pack XR‐ODS C₁₈ column with gradient elution of water (0.1% formic acid) and acetonitrile (0.1% formic acid) solution was used for separation at a flow rate of 0.3 mL/min. The calibration curves of two analytes in serum showed excellent linearity over the concentration ranges of 0.03–10 μg/mL for cefoperazone, and 0.01–3 μg/mL for sulbactam, respectively. This method involves simple sample preparation steps and was validated according to standard US Food and Drug Administration and European Medicines Agency guidelines in terms of selectivity, linearity, detection limits, matrix effects, accuracy, precision, recovery and stability. This assay can be easily implemented in clinical practice to determine concentrations of cefoperazone and sulbactam in children.     

Development and validation of a sensitive and high‐throughput LC‐MS/MS method for the simultaneous determination of esomeprazole and naproxen in human plasma

A sensitive and high‐throughput LC‐MS/MS method has been developed and validated for the combined determination of esomeprazole and naproxen in human plasma with ibuprofen as internal standard. Solid‐phase extraction was used to extract both analytes and internal standard from human plasma. Chromatographic separation was achieved in 4.0 min on XBridge C₁₈ column using acetonitrile–25 mM ammonium formate (70:30, v/v) as mobile phase. Mass detection was achieved by ESI/MS/MS in negative ion mode, monitoring at m/z 344.19 → 194.12, 229.12 → 169.05 and 205.13 → 161.07 for esomeprazole, naproxen and IS, respectively. The calibration curves were linear from 3.00 to 700.02 ng/mL for esomeprazole and 0.50 to 150.08 ng/mL for naproxen. The intra‐ and inter‐batch precision and accuracy across four quality control levels met established criteria of US Food and Drug Administration guidelines. The assay is suitable for measuring accurate esomeprazole and naproxen plasma concentrations in human bioequivalence study following combined administration. Copyright © 2013 John Wiley & Sons, Ltd.     

LC‐MS/MS analysis of Δ9‐tetrahydrocannabinolic acid A in serum after protein precipitation using an in‐house synthesized deuterated internal standard

An assay based on liquid chromatography/tandem mass spectrometry is presented for the fast, precise and sensitive quantitation of Δ9‐tetrahydrocannabinolic acid A (THCA) in serum. THCA is the biogenetic precursor of Δ9‐tetrahydrocannabinol in cannabis and has aroused interest in the pharmacological and forensic field especially as a potential marker for recent cannabis use. After addition of deuterated THCA, synthesized from D₃‐THC as starting material, and protein precipitation, the analytes were separated using gradient elution on a Luna C18 column (150 × 2.0 mm × 5 µm) with 0.1% formic acid and acetonitrile/0.1% formic acid. Data acquisition was performed on a triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode with negative electrospray ionization. After optimization, the following sample preparation procedure was used: 200 μL serum was spiked with internal standard solution and methanol and then precipitated ‘in fractions’ with 500 μL ice‐cold acetonitrile. After storage and centrifugation, the supernatant was evaporated and the residue redissolved in mobile phase. The assay was fully validated according to international guidelines including, for the first time, the assessment of matrix effects and stability experiments. Limit of detection was 0.1 ng/mL, and limit of quantification was 1.0 ng/mL. The method was found to be selective and proved to be linear over a range of 1.0 to 100 ng/mL using a 1/x weighted calibration model with regression coefficients >0.9996. Accuracy and precision data were within the required limits (RSD ≤ 8.6%, bias: 2.4 to 11.4%), extractive yield was greater than 84%. The analytes were stable in serum samples after three freeze/thaw cycles and storage at ?20 °C for one month. Copyright © 2012 John Wiley & Sons, Ltd.     

High‐performance liquid chromatography tandem mass spectrometry method for the determination of 2CC‐NBOMe and 25I‐NBOMe in human serum

2CC‐NBOMe {4‐chloro‐2,5‐dimethoxyphenethyl‐N‐[(2‐methoxyphenyl) methyl] ethanamine} and 25I‐NBOMe {2‐(4‐iodo‐2,5‐dimethoxyphenyl)‐N‐[(2‐methoxyphenyl) methyl] ethanamine} are of a class of N‐benzyl phenethylamine derivatives whose synthesis was first reported in the scientific literature in 2011. Recent reports from ‘personal drug experience websites’ and in the popular press indicate these drugs are the latest in a series of designer ‘Bath Salt’ drugs of abuse. The presented high‐performance liquid chromatography triple quadrupole mass spectrometry (HPLC/MS/MS) method was developed for the detection and quantification of 2CC‐NBOMe and 25I‐NBOMe in serum of intoxicated emergency department patients. The assay applies 2‐?(2,?5‐?dimethoxyphenyl)‐?N‐?(2‐?methoxybenzyl) ethanamine (25H‐NBOMe) as the internal standard. Samples were extracted using solid‐phase extraction columns. The chromatographic separation was performed on a Luna 3 µ C₈(2) 100 Å, 100 × 2.0 mm, column. Detection was accomplished by multiple‐reaction monitoring via an electrospray ionization source operating in the positive ionization mode. The calibration curves were linear over the investigated concentration range, 30–2000 pg/mL, with a lower limit of detection of 10 pg/mL for both 2CC‐NBOMe and 25I‐NBOMe. The method proved suitable for serum clinical toxicology testing. Two severely intoxicated emergency department patients were determined to have serum concentrations of 250 and 2780 pg/mL of 25I‐NBOMe using the presented method. Copyright © 2013 John Wiley & Sons, Ltd.     

Bioanalysis of tolvaptan,a novel AVP‐V2 receptor antagonist in human plasma by a novel LC‐ESI‐MS/MS method: a pharmacokinetic application in healthy South Indian male subjects

A simple, rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) assay method is proposed for the determination of tolvaptan in human plasma samples using tolvaptan d₇ as internal standard (IS). Analyte and the IS were extracted from 100 μL of human plasma via simple liquid–liquid extraction. The chromatographic separation was achieved on a C₁₈ column using a mixture of methanol and 0.1% formic acid buffer (80:20, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.05–501 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The intra‐day and inter‐day precision (coefficient of variation) and accuracy results in three validation batches across five concentration levels were well within the acceptance limits. A run time of 2.0 min for each sample made it possible to analyze more samples in a short time, thus increasing the productivity. The proposed method was successfully applied to a pharmacokinetic study of 15 mg and 60 mg tolvaptan tablet formulation in healthy South Indian male subjects under fasting condition. Copyright © 2013 John Wiley & Sons, Ltd.     

Immuno‐capture as ultimate sample cleanup in LC‐MS/MS determination of the early stage biomarker ProGRP

This paper presents a selective and efficient sample preparation procedure for NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker ProGRP, in human serum. The procedure is based on immuno‐capture of ProGRP in 96‐wells microtiter plates coated with the mAb E146. After immuno‐capture and thorough rinse, trypsin was added for in‐well digestion. Subsequently the signature peptide was enriched by SPE and determined by LC‐MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole MS was used during optimization of the method. A triple quadropole MS was used in the method evaluation in order to improve sensitivity. The evaluation showed good repeatability (RSD, 11.9–17.5%), accuracy (3.0–6.6%), and linearity (r² = 0.995) in the tested range (0.5–50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and 0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was compared to a previous published LC‐MS method for the determination of ProGRP in serum based on protein precipitation and online sample cleanup. Both showed acceptable method performance, however, the immuno‐capture LC‐MS method was superior with respect to sensitivity. This illustrates the large potential of immuno‐capture sample preparation prior to LC‐MS in protein biomarker quantification.     

Use of a small particle solid‐core packing for improved efficiency and rapid measurement of sirolimus and everolimus by LC‐MS/MS

Measurement of whole blood sirolimus and everolimus is required in order to optimize patient treatment following solid organ transplant. Assay by LC‐MS/MS is increasingly preferred; however efficient use of the instrument and short turnaround times are crucial. Use of a 1.6 µm solid‐core packing HPLC column (Cortecs) gave significant increases in efficiency, sensitivity and throughput compared with an existing method, following simple protein precipitation of small‐volume (20 μL) whole blood samples. Sirolimus, everolimus and the stable isotopic internal standard (¹³C₂D₄ – everolimus) eluted at around 0.8 min, and total analytical run time was 2.2 min, saving almost 4 min per sample compared with an existing method. Within‐assay imprecision (CV) was 3.3–8.5%, and between‐assay imprecision was 2.2–10.8%. Retrospective assay of external quality assurance samples and comparison of patient samples assayed in parallel showed only small differences (between +6.8 and ?1.9%) in results using the Cortecs column when compared with the existing method. No significant interferences or ion suppression were observed. Copyright © 2015 John Wiley & Sons, Ltd.