Inter-day Relative Error Research Articles

Quantitative assessment of daratumumab in serum via intact light chain measurement using liquid chromatography-high resolution mass spectrometry: a method suitable for therapeutic drug monitoring.

Daratumumab, a pivotal treatment for multiple myeloma, exhibits considerable inter-patient variability in pharmacological clinical outcomes, likely attributed to serum concentration that may underscore the need for its therapeutic drug monitoring. This study aims to develop and validate a straightforward analytical method for quantifying daratumumab in serum, focusing on intact light chain determination, using liquid chromatography high-resolution mass spectrometry. The sample preparation involved immunoglobulin enrichment using Melon gel followed by a reduction step to dissociate the light from the heavy chains of immunoglobulins. The latter were then separated using a MabPac RP 2.1 × 50 mm chromatographic column and the intact light chains were detected and quantified using a Q Exactive Orbitrap mass spectrometer operating in ESI-positive ion mode at 17 500 resolution. The method demonstrated excellent linearity (R2 > 0.992) across a serum concentration range of 100 to 2000 μg mL-1 and good precision and accuracy: intra- and interday relative errors ranged from -5.1% to 6.5%, with a relative standard deviation of less than 5.8%. Clinical suitability was confirmed by analyzing 80 clinical samples from multiple myeloma patients treated with 1800 mg of daratumumab. 99% of the samples fell within the analytical range with a mean daratumumab concentration evaluated before the next administration (Ctrough) of 398 μg mL-1. These findings highlighted that intact light chain monoclonal antibody quantification could be a valid and robust alternative to either immunoassays or to LC-MS/MS targeting peptides for measuring daratumumab in clinical samples, positioning it as a suitable method for therapeutic drug monitoring applications.

Simultaneous quantification of mirabegron and vibegron in human plasma by HPLC-MS/MS and its application in the clinical determination in patients with tumors associated with overactive bladder

Mirabegron and vibegron, both newly identified beta-3 adrenergic agonists, have significantly improved the quality of life for patients suffering from overactive bladder. In order to comprehensively assess the plasma exposure levels of these agents, the development of a rapid and highly sensitive bioanalytical method becomes imperative. The primary objective of this study was to establish a robust high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the concurrent quantification of mirabegron and vibegron in human plasma. The analytes were extracted from a 100 μL plasma sample through protein precipitation, employing 300 μL of methanol. Subsequently, samples underwent separation and quantification using a Waters XBridge C18 column (2.1 × 100 mm, 3.5 µm), with a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The mass analysis was conducted using positive electrospray ionization (ESI+) operated in a multiple reaction monitoring (MRM) mode. The proposed method was meticulously validated in accordance with the guidelines set forth by the U.S. Food and Drug Administration (FDA) for bioanalytical method validation. The regression equations demonstrated exceptional linearity for both mirabegron (r² ≥ 0.994) and vibegron (r² ≥ 0.996) across the concentration range of 0.5 – 200 ng/mL. Furthermore, the assay exhibited accuracy (inter-day relative error ≤ 6.90%) and precision (inter-day coefficient of variation ≤ 8.88%). The average recoveries of the analytes were found to range from 81.94% to 102.02%, with mean matrix effects falling within the range of 89.77% to 110.58%. As a result, this method was deemed highly suitable for the precise determination of the concentrations of both mirabegron and vibegron in the context of therapeutic drug monitoring and bioequivalence studies.

Pharmacokinetics, bioavailability and tissue distribution of chitobiose and chitotriose in rats

Chitooligosaccharides (COSs) have various physiological activities and broad application prospects; however, their pharmacokinetics and tissue distribution remain unclear. In this study, a sensitive and selective ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method for determining chitobiose (COS 2) and chitotriose (COS 3) in rat serum and tissues was developed. This method was successfully validated based on FDA guidelines in terms of selectivity, calibration curves (lower limit of quantification was 0.002 µg/mL for COS 2 and 0.02 µg/mL for COS 3), precision (intra-day relative standard deviation of 0.04%–3.55% and inter-day relative standard deviation of 1.94%–11.63%), accuracy (intra-day relative error of − 1.81%–11.06% and inter-day relative error of − 9.41%–8.63%), matrix effects, recovery (97.10%–101.29%), stability, dilution integrity, and carry-over effects. Then, the method was successfully applied to the pharmacokinetics and tissue distribution study of COS 2 and COS 3 after intragastric and intravenous administration. After intragastric administration, COS 2 and COS 3 were rapidly absorbed, reached peak concentrations in the serum after approximately 0.45 h, and showed rapid elimination with clearances greater than 18.82 L/h/kg and half-lives lower than 6 h. The absolute oral bioavailability of COS 2 and COS 3 was 0.32%–0.52%. COS 2 and COS 3 were widely distributed in Wistar rat tissues and could penetrated the blood–brain barrier without tissue accumulation.Graphic

Quantitation of Total Vanadium in Rodent Plasma and Urine by Inductively Coupled Plasma – Mass Spectrometry (ICP-MS)

Human exposure to vanadium (V) is anticipated because it is a drinking water contaminant. Due to limited data on soluble V salts, the National Toxicology Program is investigating the toxicity in rodents following drinking water exposure. Measurement of internal V dose allows for interpretation of toxicology data. The objective of this study was to develop and validate an inductively coupled plasma-mass spectrometric method to quantitate total V in rat plasma. The method was linear (r ≥ 0.99) from 5.00 − 1,000 ng V/mL. Intra- and inter-day relative error (% RE) and relative standard deviation (% RSD) of spiked plasma samples were 8.5% to 15.6% RE and ≤ 1.8% RSD and 7.3% to 11.7% RE and ≤ 3.1% RSD, respectively. The limit of detection was 0.268 ng V/mL plasma and absolute percent recovery was 113%. Standards up to 7,500 ng V/mL plasma were diluted into the validated range (5.6% RE, 0.9% RSD). V in extracted plasma samples over 15 days at ambient and refrigerated conditions was from 97.7 to 126% of day 0. Determined plasma V concentrations after three freeze-thaw cycles and after frozen storage for up to 63 days ranged from 100 to 106% and 100 to 122% of day 0, respectively. The method was extended to rat urine (accuracy and precision −2.0 to 0.3% RE and <0.6% RSD, respectively for same linear range). These data demonstrate that the method is suitable to quantitate V in rat plasma and urine.

In-tube stir bar sorptive extraction based on 3-aminopropyl triethoxysilane surface-modified Ce-doped ZnAl layered double hydroxide thin film for determination of nonsteroidal anti-inflammatory drugs in saliva samples

Athin-film based on 3-aminopropyl triethoxysilane surface-modified Ce-doped zinc-aluminum layered double hydroxide was synthesized on the inner surface of an aluminum tube. It has beenapplied toin-tube stir bar sorptive extraction of nonsteroidal anti-inflammatory drugs in saliva samples followed by high-performance liquid chromatography. The sorbent was characterized by scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and elemental mapping. The extraction parameters including sample pH (4.2), extraction time (10min), stirring speed (800rpm), type of eluent (acidified tetrahydrofuran), eluent volume (100μL), and desorption time (6min) were thoroughly optimized. Under the optimum conditions, limits of detection were found to be less than 5.0ngmL-1. Calibration plots were linear within the range 10-1000ngmL-1 (R2 > 0.9982). Absolute recoveries were calculated in the range 63.5 to 72.4%. The repeatability (intra- and inter-day precision) and reproducibility (tube-to-tube precision) at concentrations of 50, 250, and 500ngmL-1 were less than 7.6% and 9.4%, respectively. The method accuracy based on the relative error was calculated at these concentrations and ranged from - 4.9 to - 9.3% for intra-day relative error (%) and - 6.8 to - 11% for inter-day relative error (%). Finally, the method applicability was examined for the determination of nonsteroidal anti-inflammatory drugs in saliva samples, and good relative recoveries were obtained within the range 86.5 to 95.2%. As a result, the introduced method can be applied as a suitable alternative to measuring nonsteroidal anti-inflammatory drugs in biological fluids. Graphical abstract A surface-modified Ce-doped ZnAl LDH thin film was synthesized on the inner surface of an Al tube and applied for in-tube stir bar sorptive extraction of NSAIDs in saliva.

Determination of sirolimus in blood by high-performance liquid chromatography with ultraviolet detection - Experience and review

Sirolimus (SRL) is a potent immunosuppressant. Therapeutic drug monitoring (TDM) of SRL is required to optimize the therapy. Immunoassay, high-performance liquid chromatography with ultraviolet detection (HPLC-UV), and HPLC with mass-spectrometric detection (HPLC-MS or HPLC/MS/MS) have been used in the analysis of SRL. The purpose of this study was to share our experience in validating a HPLC-UV method for analyzing SRL and to have an overview of HPLC-UV methods used in SRL assay. A validated HPLC/UV method developed by Wyeth-Ayerst Research with minor modification was use to determine SRL concentration in human whole blood. An Alltima C18 column (5 μm, 150 × 2.1 mm) was used as the stationary phase. The mobile phase was 60% acetonitrile in water, and the flow rate was 0.5 mL/min. Samples were prepared by spiking human whole blood (0.5 mL) with the internal standard (IS) and designated amount of SRL, except blank. Zinc sulfate (50 g/L, 1 mL) and acetone (1 mL) were used for hemolysis and deproteinization. After alkalizing supernatant with 100 mM NaOH (0.2 or 0.3 mL), 1-chlorobutane (2 mL) was used for extraction. The 1-chlorobutane layer was dried, reconstituted with mobile phase and back extracted with 0.5 mL of n-hexane. The limitation of quantification was 2.5 ng/mL and the standard curve was linear at the concentration range of 2.5-75 ng/mL. The intraday and interday coefficients of variation were 2.1-5.2% and 2.8-5.7%, respectively. The intraday and interday relative errors were -0.03-5.3% and 0.7-3.3%, respectively. The recoveries for SRL and the IS were 78.5-92.8% and 76.9 ± 3.9%, respectively. The samples were proven to be stable after 3 freeze/thaw cycles, and the extract was stable over 24 hr at an autosampler set to 4°C. In addition to an overview of the chemical properties of SRL, different HPLC-UV methods for the quantification of SRL were provided to identify analytic parameters that are critical for the establishment of HPLC-UV assay for SRL.

A rapid HPLC–MS/MS method for determining busulfan in hemolytic samples from children with hematopoietic stem cell transplantation

A rapid and sensitive method for the quantitative detection of busulfan (BU) in children's hemolytic samples by HPLC-tandem mass spectrometry (MS/MS) was established. In this study, the sample preparation procedure involved a one-step protein precipitation with acetonitrile (ACN) solution, and the HPLC-MS/MS method used Hypersil GOLD C18 . The mobile phase consisted of 10 mM ammonium acetate solution (containing 0.1% formic acid) and ACN with a flow rate of 0.4 mL/min. Multiple reaction monitoring modes were used for quantitative analysis and the ion pairs of BU and BU-d8 were m/z 263.9 → 150.9 and 272.0 → 159.0, respectively. BU had a good linearity in the range of 0.01-10 μg mL-1 . The intra- and inter-day relative error was between -7.21% and 8.26%, and the coefficient of variation was less than 12.64%. The average extraction recovery rate in plasma samples was 99.76% ± 6.53%, and the matrix in normal plasma and hemolyzed plasma had no significant effect on the detection results. Normal and hemolytic samples could maintain good stability at 4, 25 and -40°C. As a result, this method is particularly suitable for determining BU in hemolytic samples from children with hematopoietic stem cell transplantation (HSCT), and this study provides the methodological basis for further research on the pharmacokinetics of BU in children with HSCT.

Novel method for fast trabectedin quantification using hydrophilic interaction liquid chromatography and tandem mass spectrometry for human pharmacokinetic studies

Few time-consuming bioanalytical methods are currently available for trabectedin quantification in clinical investigations. Here we present a novel, fast and sensitive method for trabectedin determination in human plasma based on hydrophilic interaction liquid chromatography and tandem mass spectrometry (HILIC-MS/MS). Plasma samples are treated with acetonitrile–0.1 % formic acid and the solvent extract is directly injected into an Acquity BEH Amide column (2.1 × 100 mm, 1.7 μm) operating in HILIC mode at 0.2 mL/min with 80:20 acetonitrile–0.1 % formic acid in water. The analyte is separated by an organic solvent gradient and quantified by an Agilent Ultivo triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode. The quantitative MRM transitions were m/z 762→234 and m/z 765→234 for trabectedin and its d3-labeled derivative, respectively. The lower limit of quantification (LLOQ) was 0.01 ng/mL and the assay was linear up to 2.5 ng/mL. The intra- and inter-day relative error ranged from 1.19 % to 8.52 %, while the relative standard deviation was less than 12.35 %. The method was used to determine the pharmacokinetic profiles of trabectedin in 26 patients with soft tissue sarcoma, showing that this new HILIC-MS/MS method is suitable for use in clinical research.

Development and Validation of an Analytical Method to Quantitate Tris(chloroisopropyl)phosphate in Rat and Mouse Plasma using Gas Chromatography with Flame Photometric Detection.

Tris(chloropropyl)phosphate (TCPP) is an organophosphorus flame retardant (OPFR) and plasticizer increasingly used in consumer products and as a replacement for brominated flame retardants. Commercially available TCPP is a mixture of four structural isomers the most abundant of which is tris(1-chloro-2-propyl)phosphate (TCPP-1). Although there is a widespread use of TCPP and potential for human exposure, there is limited data on the safety or toxicity of TCPP. The National Toxicology Program is conducting long-term studies to examine the toxicity of the TCPP in rats after lifetime exposure, including perinatal oral exposure. Quantitative estimates of internal dose are essential to interpret toxicological findings in rodents. To aid in this, a method was fully validated to quantitate the most abundant isomer, TCPP-1, in female Harlan Sprague Dawley (HSD) rat and B6C3F1 mouse plasma with partial validation in male rat plasma, and male and female mouse plasma. The method used protein precipitation using trichloroacetic acid followed by the extraction with toluene, and analysis by gas chromatography with flame photometric detection. The performance of the method was evaluated over 5-70 ng TCPP-1/mL plasma. The method was linear (r ≥ 0.99), accurate (inter-day relative error: ≤ ± -7.2) and precise (inter-batch relative standard deviation: ≤27.5%). The validated method has lower limits of quantitation and detection of ~5 and 0.9 ng/mL, respectively, in female HSD rat plasma and can be used on samples as small as 50 μL demonstrating the applicability to plasma samples from toxicology studies.

Simultaneous quantification of phencynonate and its active metabolite N-demethyl phencynonate in human plasma using liquid chromatography and isotope-dilution mass spectrometry.

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously quantify phencynonate (PCN) and its major metabolite N-demethyl phencynonate (DM-PCN) in human plasma. Following one-step liquid-liquid extraction, the analytes were separated on a reversed-phase C18 column. Methanol and 0.02% formic acid in 10 mM ammonium acetate (62:38, v/v) was used as isocratic mobile phase at a flow-rate of 0.3 mL/min. An API 5000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. Multiple reaction monitoring using the transition of m/z 358.4 → m/z 156.2, m/z 344.4 → m/z 142.2, and m/z 361.3 → m/z 159.2 was performed to quantify PCN, DM-PCN, and the internal standard (D3 -PCN), respectively. This approach showed a lower limit of quantification of 10 pg/mL and 25 pg/mL for PCN and DM-PCN in plasma, respectively. This sensitivity was at least 50-fold superior to previously reported ones and thus enabled the approach well applicable to low-dose pharmacokinetic studies. The intra- and inter-day precisions were less than 14.2 % at each QC level for both PCN and DM-PCN. The inter-day relative errors ranged from -1.9% to -4.9% for PCN, and from 0.6% to 6.4% for DM-PCN. As a proof of principle, the validated method was successfully applied to simultaneous quantification of circulating PCN and DM-PCN in healthy subjects after a single oral administration of 2 mg phencynonate hydrochloride pellet.

Micro-method for the determination of glutathione in human blood

A new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5 μL) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of haemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5'-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artefactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with -1.4% to 2.3% intra-day relative error (RE) and -2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood.

Determination of Rhodamine 123 in rat plasma utilizing liquid chromatography–tandem mass spectrometry

Rhodamine 123 (R123), as a typical of P-gp substrate, was widely used to quantify P-glycoprotein (P-gp) functional efflux activity in vivo. A new, rapid and sensitive method was developed for quantifying R123 in rat plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS). R123 and Rhodamine 6G (R6G, the internal standard, IS) were extracted from aliquots of plasma with ethyl acetate and dichloromethane (4:1) as the solvent and chromatographic separation was performed using a Zorbax Eclipse Plus C18 column. The mobile phase was composed of A: ammonium formate–formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid and B: methanol (A:B, 5:95, v/v). To quantify R123 and IS respectively, multiple reaction monitoring (MRM) transition of m/ z 345.2 → 285.2 and m/ z 443.3 → 415.2 was performed. The analysis time was 4 min in positive mode; the calibration curve was linear in the concentration range of 1–200 ng/ml. The lowest limit of quantification (LLOQ) reached 1 ng/ml. The intra and inter-day precision were less than 9.2% for the low quality control (QC) level, and 3.4% for other QC levels, respectively, while the intra and inter-day relative errors ranged between −7.4% and 9.1% for three QC concentration levels. The LC–MS/MS method proved to be simple, accurate, reliable and with a shorter running time and has been successfully applied to evaluate the functional activity of P-glycoprotein in an absorption experiment in the rat.

Simultaneous determination of selenomethionine enantiomers in biological fluids by stable isotope dilution gas chromatography–mass spectrometry

A method for the stereoselective determination of d- and l-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography–mass spectrometry with selected-ion monitoring (GC–MS-SIM). dl-[ 2H 3, 82Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for d- and l-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for d-selenomethionine and 6% and 3% for l-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for d-selenomethionine and 2% and 1% for l-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.

Determination of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry

A sensitive and rapid method was developed for quantification of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry (LC–MS/MS). An aliquot of 1 mL plasma sample was extracted with ethyl acetate–dichloromethane. Separation of olprinone and the milrinone (internal standard, IS) from the interferences was achieved on a C 18 column followed by MS/MS detection. The analytes were monitored in the positive ionization mode. Multiple reaction monitoring using the transition of m/ z 251 → m/ z 155 and m/ z 212 → m/ z 140 was performed to quantify olprinone and IS, respectively. The method had a total chromatographic run time of 3 min and linear calibration curves over the concentration range of 0.5–60 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. The intra- and inter-day precisions were less than 16.3% for low QC level, and 7.1% for other QC levels, respectively. The intra- and inter-day relative errors were ranged between −12.2% and 3.7% for three QC concentration levels. The validated method was successfully applied to the quantification of olprinone concentration in human plasma after intravenous (i.v.) administration of olprinone at a constant rate of infusion of 2 μg/(kg min) for 5 min in order to evaluate the pharmacokinetics.

Determination of eptifibatide concentration in human plasma utilizing the liquid chromatography–tandem mass spectrometry method

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to determine the concentration of eptifibatide in human plasma. Following protein precipitation, the analyte was separated on a reversed-phase C 18 column. Acetonitrile:5 mM ammonium acetate:acetic acid (30:70:0.1, v/v/v) was used at a flow-rate of 0.5 mL/min with the isocratic mobile phase. An API 4000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. “Truncated” multiple reaction monitoring using the transition of m/ z 832.6 → m/ z 832.6 and m/ z 931.3 → m/ z 931.3 was performed to quantify eptifibatide and the internal standard (EPM-05), respectively. The method had a lower limit of quantification of 4.61 ng/mL for eptifibatide. The calibration curve was demonstrated to be linear over the concentration range of 4.61 − 2770 ng/mL. The intra- and inter-day precisions were less than 10.5% for each QC level, and the inter-day relative errors were 2.0%, 5.6%, and 2.8% for 9.22, 184, and 2490 ng/mL, respectively. The validated method was successfully applied to the quantification of eptifibatide concentration in human plasma after intravenous (i.v.) administration of a 270-μg/kg bolus of eptifibatide and i.v. administration of eptifibatide at a constant rate of infusion of 2 μg/(kg min) for 18 h in order to evaluate the pharmacokinetics.

Quantitative determination of tilmicosin in canine serum by high performance liquid chromatography–tandem mass spectrometry

A highly sensitive and quantitative LC/MS/MS assay for the determination of tilmicosin in serum has been developed and validated. For sample preparation, 0.2 mL of canine serum was extracted with 3 mL of methyl tert-butyl ether. The organic layer was transferred to a new vessel and dried under nitrogen. The sample was then reconstituted for analysis by high performance liquid chromatography–tandem mass spectrometry. A Phenomenex Luna C8(2) analytical column was used for the chromatographic separation. The eluent was subsequently introduced to the mass spectrometer by electrospray ionization. A single range was validated for 50–5000 ng/mL for support of toxicokinetic studies. The inter-day relative error (inaccuracy) for the LLOQ samples ranged from −5.5% to 0.3%. The inter-day relative standard deviations (imprecision) at the respective LLOQ levels were ≤10.1%.

Simultaneous determination of methylephedrine and noscapine in human plasma by liquid chromatography–tandem mass spectrometry

A selective and sensitive method has been developed and validated for simultaneous quantification of methylephedrine and noscapine in human plasma. Analytes were extracted from human plasma samples by liquid–liquid extraction, separated on a Diamonsil C 18 column and detected by tandem mass spectrometer with an atmospheric pressure chemical ionization (APCI) interface. Diphenhydramine was used as the internal standard (I.S.). The method was found to be precise and accurate within the linear range 0.1–100 ng/ml for each analyte. The intra- and inter-day relative standard deviations (R.S.D.s) were below 5.2% for methylephedrine and 6.7% for noscapine. The inter-day relative error (RE) as determined from quality control samples (QCs) was less than 3.0% for each analyte. The assay was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation containing 20 mg dl-methylephedrine hydrochloride, 16 mg noscapine, 300 mg paracetamol and 1 mg of chlorpheniramine maleate.

Study on the bioavailability of nateglinide-hydroxypropyl-beta-cyclodextrin complex capsule in rabbits by liquid chromatographic-tandem mass spectrometry.

To evaluate the bioavailability of nateglinide-hydroxypropyl-beta-cyclodextrin (HPCD) complex, a rapid and specific liquid chromatographic-tandem mass spectrometric method was developed and validated to determine nateglinide in rabbit serum. The analyte was extracted from serum samples by liquid-liquid extraction, separated on a Zorbax C(18) column and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface. Daidzein was used as the internal standard. The method has a lower limit of quantitation of 0.25 mg/L using 200 micro L serum. The intra- and inter-day relative standard deviations calculated from quality control (QC) samples were below 4%. The inter-day relative error was within 1%. Nateglinide serum concentrations in rabbits given nateglinide-hydroxypropyl-beta-cyclodextrin complex were much higher than those given the free drug. Significant difference was observed in main pharmacokinetic parameters of t(max) and C(max) but not AUC(0-t) between the complex and free drug. It was concluded that the absorption rate of nateglinide-HPCD complex was enhanced, compared with that of nateglinide free drug.

Simultaneous determination of chlorpheniramine and pseudoephedrine in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and specific procedure for simultaneous quantitation of chlorpheniramine and pseudoephedrine in human plasma has been developed and validated. Analytes were extracted from plasma samples by liquid-liquid extraction, separated on a Diamonsil C18 column (250 x 4.6 mm i.d.) and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface. Diphenhydramine was used as the internal standard. The method has a lower limit of quantitation of 0.2 and 2.0 ng/mL for chlorpheniramine and pseudoephedrine, respectively. The intra- and inter-day relative standard deviation, calculated from quality control (QC) samples were below 4.3% for chlorpheniramine and below 9.5% for pseudoephedrine. The inter-day relative error as determined from QC samples was within 4.7% for each analyte. The overall extraction recoveries of chlorpheniramine and pseudoephedrine were 77 and 61% on average, respectively. The method was successfully applied to pharmaockinetic study of chlorpheniramine and pseudoephedrine in volunteers receiving formulations containing 4 mg of chlorpheniramine maleate and 60 mg of pseudoephedrine hydrochloride.

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [ 2H 7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6- 2H 7]α-ketoisocaproic acid ([ 2H 7]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5- 2H 3]α-Ketoisocaproic acid ([ 2H 3]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [ 2H 3]KIC and [ 2H 7]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection ( S/N=3) and the limit of quantitation for [ 2H 7]KIC was around 50 n M in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [ 2H 7]KIC spiked to rat plasma in the range of 0.1 to 10 μ M gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [ 2H 7]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [ 2H 7]KIC after an intravenous administration of [ 2H 7]KIC in rat.