Validated LC–MS–MS Method for Quantitative Determination of Batifiban in Human Plasma and Its Application to a Pharmacokinetic Study

Batifiban is a new platelet GPIIb/IIIa receptor antagonist. In this work, an analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry has been firstly developed and validated for the quantitative measurement of batifiban in human plasma to support the investigation of this compound. Separation of analyte and the internal standard eptifibatide was performed on a Thermo HyPURITY C18 column (150 × 2.1 mm, 5 μm) with a mobile phase consisting of formic acid 0.1% (v/v)–acetonitrile (40:60, v/v) at a flow rate of 0.25 mL min^?1. The Waters QuattroMicro API triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode via positive electrospray ionization interface using the transition m/z 819.2 → m/z (623.9 + 159.4) for batifiban and m/z 833.4 → m/z (645.7 + 159.3) for IS. The method was linear over the concentration range of 2.45–5,000 μg L^?1. The intra- and inter- day precisions were less than 15% in terms of relative standard deviation, and the accuracy was within 8.5% in terms of relative error (RE). The lower limit of quantification (LLOQ) was identifiable and reproducible at 2.45 μg L^?1 with acceptable precision and accuracy. The validated method offered sensitivity and wide linear concentration range. This method was successfully applied for the evaluation of pharmacokinetics of batifiban afer single oral doses of 55, 110 and 220 μg kg^?1 batifiban to 36 Chinese healthy volunteers.     

Determination of Mildronate in Human Plasma and Urine by UPLC�CPositive Ion Electrospray Tandem Mass Spectrometry

A simple, rapid, and selective method to determine the concentration of mildronate in human plasma and urine using ultra performance liquid chromatography?Ctandem mass spectrometry (UPLC-MS-MS) was developed and validated. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization at m/z 147.2?C58.0 for mildronate and m/z 147.2?C87.8 for the internal standard, carbachol. The UPLC separation was carried out with a UPLC BEH HILIC column. The mobile phase consisted of 0.08% formic acid in 30 mM ammonium acetate solution and acetonitrile (23:77, v/v). Plasma samples were extracted from plasma by protein precipitation and urine samples were diluted with the mobile phase. The analysis time was 3.5 min for each sample. Linear calibration curves ranged from 0.10 to 100.00 ??g mL^?1 in human plasma and 0.50 to 600.00 ??g mL^?1 in urine. The method had been successfully applied to a pharmacokinetic study in healthy volunteers. After single intravenously administration of 250, 500, and 750 mg mildronate, the elimination half-life (t _1/2) were (2.74 ± 0.67), (4.86 ± 0.82) and (5.16 ± 0.77) h, respectively. The t _1/2 for the 250 mg dose did vary significantly with other dosages (P < 0.05), mildronate may have non-linear pharmacokinetics in humans.     

Clustering and Filtering Tandem Mass Spectra Acquired in Data-Independent Mode

Data-independent mass spectrometry activates all ion species isolated within a given mass-to-charge window (m/z) regardless of their abundance. This acquisition strategy overcomes the traditional data-dependent ion selection boosting data reproducibility and sensitivity. However, several tandem mass (MS/MS) spectra of the same precursor ion are acquired during chromatographic elution resulting in large data redundancy. Also, the significant number of chimeric spectra and the absence of accurate precursor ion masses hamper peptide identification. Here, we describe an algorithm to preprocess data-independent MS/MS spectra by filtering out noise peaks and clustering the spectra according to both the chromatographic elution profiles and the spectral similarity. In addition, we developed an approach to estimate the m/z value of precursor ions from clustered MS/MS spectra in order to improve database search performance. Data acquired using a small 3 m/z units precursor mass window and multiple injections to cover a m/z range of 400–1400 was processed with our algorithm. It showed an improvement in the number of both peptide and protein identifications by 8 % while reducing the number of submitted spectra by 18 % and the number of peaks by 55 %. We conclude that our clustering method is a valid approach for data analysis of these data-independent fragmentation spectra. The software including the source code is available for the scientific community.

Determination of Dydrogesterone in Human Plasma by Tandem Mass Spectrometry: Application to Therapeutic Drug Monitoring of Dydrogesterone in Gynecological Disorders

A sensitive and specific tandem mass spectrometric (MS–MS) method was developed and validated for the determination of dydrogesterone (Duphaston^®), an orally active synthetic progestogen, in human plasma. Multiple reaction monitoring (MRM) scans at m/z 313.1 > 105.5 (dydrogesterone) and m/z 393 > 147 (dexamethasone, internal standard) were selected to determine dydrogesterone by the internal standard method. Linear correlations (r: ～0.99 ± 0.05) of the calibration curves were established over the concentration range 10–60 ng mL^?1 with a lower limit of quantification (LLQ) of 10 ng mL^?1 (RSD% 14.9 and %DEVs ?10.5 to +15.6). Solid-phase extraction (SPE) technique was used for extraction of dydrogesterone and internal standard from patient plasma samples using Oasis^® Max C18 cartridges. Ion suppression studies indicated negligible effects of plasma matrix on the mass ions detection of dydrogesterone and IS, when measured in MRM mode. Validation data showed that RSD% values were <22.0%, whereas %DEV values were in the range of ?20.2 to +13.3 for intra- and inter-day precision and accuracy, respectively. Analytical recoveries of dydrogesterone from supplemented plasma samples with the drug were in the range of 100.7–112%, indicating the efficiency of the SPE for separation of dydrogesterone from human plasma. Stability studies conducted at ?20 °C, showed that dydrogesterone was stable in plasma as indicated from the measured degradation kinetic parameters. The developed method was applied for monitoring plasma levels of dydrogesterone in 25 patients treated with Duphaston^® tablets at a dose of 10 mg three times daily. Mean plasma concentration of 16.1 ± 3.5 ng mL^?1 of dydrogesterone were measured at the steady state. The data suggest the utility of tandem mass method in therapeutic drug monitoring of plasma levels of dydrogesterone in gynecological disorders treated with Duphaston^® tablets.     

Calibration Point for Electron Ionization MS / MS spectra measured with multiquadrupole mass spectrometers

A protocol for establishing standard instrument conditions for measurement of product ion MS/MS spectra from parent ions produced by electron ionization is presented. Within this protocol, the ion at m/z 231 (C₅F₉ ⁺) from perfluorokerosene or perfluorotributylamine is selected as the parent ion and subjected to collision-induced dissociation. The relative intensities of product ions at m/z 69, 131, and 181 are monitored as a function of collision energy while keeping the target gas pressure constant within the range of 10^?4–10^?6 torr (measured), or a beam attenuation of approximately 30-70%. The collision energy at which the ion intensities for product ions at m/z 69 and 181 are equal is defined as the calibration point at that collision gas pressure; the intensity of the ion at m/z 131 is very close to this value as well. Electron ionization MS/MS spectra taken at the calibration point using two different multiquadrupole instruments show good reproducibility for several test compounds. The high degree of similarity may aid in the establishment of a MS/MS spectral library.     

Fast and accurate determination of K, Ca, and Mg in human serum by sector field ICP-MS

Electrolytes in serum are important biomarkers for skeletal and cellular health. The levels of electrolytes are monitored by measuring the Ca, Mg, K, and Na in blood serum. Many reference methods have been developed for the determination of Ca, Mg, and K in clinical measurements; however, isotope dilution thermal ionization mass spectrometry (ID-TIMS) has traditionally been the primary reference method serving as an anchor for traceability and accuracy to these secondary reference methods. The sample matrix must be separated before ID-TIMS measurements, which is a slow and tedious process that hindered the adoption of the technique in routine clinical measurements. We have developed a fast and accurate method for the determination of Ca, Mg, and K in serum by taking advantage of the higher mass resolution capability of the modern sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). Each serum sample was spiked with a mixture containing enriched ⁴⁴Ca, ²⁶Mg, and ⁴¹K, and the ⁴²Ca⁺:⁴⁴Ca⁺, ²⁴Mg⁺:²⁶Mg⁺, and ³⁹K⁺:⁴¹K⁺ ratios were measured. The Ca and Mg ratios were measured in medium resolution mode (m/Δm?≈?4 500), and the K ratio in high resolution mode (m/Δm?≈?10 000). Residual ⁴⁰Ar¹H⁺ interference was still observed but the deleterious effects of the interference were minimized by measuring the sample at K?>?100 ng g^?1. The interferences of Sr⁺⁺ at the two Ca isotopes were less than 0.25 % of the analyte signal, and they were corrected with the ⁸⁸Sr⁺ intensity by using the Sr⁺⁺:Sr⁺ ratio. The sample preparation involved only simple dilutions, and the measurement using this sample preparation approach is known as dilution-and-shoot (DNS). The DNS approach was validated with samples prepared via the traditional acid digestion approach followed by ID-SF-ICP-MS measurement. DNS and digested samples of SRM 956c were measured with ID-SF-ICP-MS for quality assurance, and the results (mean ± expanded uncertainty in mg dL^?1 unit) for Ca (DNS?=?10.14?±?0.13, digested?=?10.11?±?0.10), Mg (DNS?=?2.093?±?0.008, digested?=?2.098?±?0.007), and K (DNS?=?15.48?±?0.11, digested?=?15.50?±?0.28) were in good agreement with the certified values (Ca?=?10.17?±?0.06, Mg?=?2.084?±?0.023, K?=?15.55?±?0.13). Major sources of uncertainty are sample measurement, spike calibration, and instrument factor including mass discrimination of the spectrometer and the detector deadtime.     

A multivariate calibration procedure for the tensammetric determination of detergents

A multivariate calibration procedure based on singular value decomposition (SVD) and the Ho-Kashyap algorithm is used for the tensammetric determination of the cationic detergents Hyamine 1622, benzalkonium chloride (BACl), N-cetyl-N,N,N-trimethylammonium bromide (CTABr) and mixtures of CTABr and BACl. The sensitivity and accuracy depend strongly on the nature of the detergent. Acceptable accuracy is obtained with a two-step calculation procedure in which calibration constants for the total concentration range of interest are used to guide the choice of a more specific set of calibration constants which are valid for a much smaller concentration span. For Hyamine 1622, concentrations in the range 5 × 10^?6?2 × 10^?4 M could be determined with an accuracy of ± 10^?6 M. For CTABr, these numbers were 3 × 10^?6?2 × 10^?4 M and ± 5 × 10^?7 M; for BACl, they were 2 × 10^?3?9 × 10^?2 g l^?1 and ± 1 × 10^?3 g l^?1. In the mixtures of CTABr and BACl, the accuracies were ± 3 × 10^?6 M and × 1 × 10^?3 g l^?1, respectively.     

Determination of Tiopronin in Human Plasma and Pharmacokinetics by LC-ESI-MS

A rapid, sensitive and specific method based on high performance liquid chromatography-electrospray ionization-mass spectrometry was developed for the determination of tiopronin in human plasma. In this study, vitamin C and dithiothreitol were used as the reducer and to release and stabilize tiopronin from dimeric mixed forms with endogenous thiols encountered during pretreatment of plasma samples. The separation was successfully achieved on an Agilent SB-Aq column packed with 5 μm C₁₈ silica, using an aqueous formic acid solution (pH 4.5–4.7), 0.5 mM tris(hydroxymethyl)aminomethane and methanol (95:5, v/v) as the mobile phase. Mass spectra were acquired in selective ion monitoring mode, using the [M ? H] ^? ions of tiopronin at m/z 162.0 and the [M ? H]^? of the internal standard sodium cyclamate at m/z 178.0, respectively. This quantitative assay was fully validated with respect to precision, repeatability and accuracy. The correlation coefficients were >0.9995 in the range 0.025–8.15 μg mL^?1 in human plasma. The mean recoveries were above 85%. The limit of quantitation was 0.012 μg mL^?1 with a relative standard deviation of inter-day and intra-day accuracy of less than 15%. This LC-ESI-MS method was also successfully applied to a pharmacokinetic study after oral administration of formulated tiopronin to healthy volunteers. The elimination half-life (T _1/2) was 21.5 ± 11.1 h.     

Traceability of measurement results in chemistry: a case study of certification of three new pharmaceutical reference materials

This paper compares the results of the studies carried out with three new candidate certified reference materials (CRM) of captopril, metronidazole, and sodium diclofenac, which are the first CRMs of these active pharmaceutical ingredients (APIs) reported in the literature. The studies were carried out according to the ISO Guides 34: 2009 and 35: 2006 and included the determination of organic, inorganic, and volatile impurities mass fractions, evaluation of homogeneity and stability under transport and storage conditions, calculation of API mass fractions by mass balance, cross-checking of obtained results, and estimation of measurement uncertainties. The certified property values and corresponding expanded uncertainties, obtained from the combined standard uncertainties multiplied by the coverage factor (k?=?2), for a confidence level of 95?%, were (995.65?±?0.93) mg/g for captopril, (998.87?±?0.15) mg/g for metronidazole, and (999.76?±?0.10) mg/g for sodium diclofenac. These new CRMs are intended to be used in assay and tests methods, including equipment calibration, method validation, as well as assignment of traceable property values and corresponding uncertainties to non-certified reference materials, with the objective to ensure metrological traceability of measurement results to the International System of Units, as well as results accuracy and comparability.     

Ion-Molecule reactions in methylamine and dimethylamine and trimethylamine systems

Fourier transform ion cyclotron resonance mass spectrometry has been used to measure the reaction rates for ions derived from methylamine with dimethylamine or trimethylamine. The use of the selective ion ejection technique greatly simplifies the elucidation of the ion-molecule reaction channels. The rate constants for proton transfer from protonated metwlamine, CH₃NH ₃ ⁺ (m/z 32), to dimethylamine and trimethylamine are 16.1 ± 1.6 × 10^?10 and 9.3 ± 0.9 × 10^?10 cm³ molec^?1s^?1, respectively. The rate constants for charge transfer from methylamine molecular ion, CH₃NH ₂ ⁺ (m/z 31), to dimethylamine and trimethylamine are 9.3 ± 1.8 x 10^?10 and 15.0 ± 5 × 10^?10 cm³molec^?1s^?1, respectively.     

Validation of an analytical LC-MS/MS method in human plasma for the pharmacokinetic study of atomoxetine

This study aimed to validate a sensitive and reliable analytical method for the pharmacokinetic study of atomoxetine in human plasma by liquid chromatography-electrospray ionization-tandem mass spectrometry. Metoprolol was used as an internal standard. After liquid-liquid extraction with methyl t-butyl ether, the supernatant was evaporated. The residue was then reconstituted and an aliquot was injected into the high performance liquid chromatographic system. Separation was performed on a Phenomenex Luna C₁₈ column (2.0 mm × 100 mm, 3 μm particles) with a mobile phase of 10 mM ammonium formate buffer: methanol = 10: 90 (v/v). Tandem mass spectrometry was performed in the electrospray ionization positive ion mode using the multiple reaction monitoring mode for quantification. The mass transition pairs of m/z 256 → 44 for atomoxetine and m/z 268 → 116 for the internal standard were used. The flow rate of the mobile phase was 0.25 mL/min and the retention times of atomoxetine and the internal standard were found to be 1.0 and 0.9 min, respectively. The calibration curve for atomoxetine was linear in the concentration range of 1–750 ng/mL (r ² = 0.9992) with a lower limit of quantification of 1 ng/mL. The mean accuracy for atomoxetine was 93–102%. The coefficients of variation (precision) in the intra- and inter-day validation for atomoxetine were 4.0–6.8 and 1.1–9.6%, respectively. The pharmacokinetic parameters of atomoxetine were evaluated after administration of a 40-mg single oral dose to twelve healthy male volunteers. The mean AUC_{0–24 h}, C _max, T _max and T _1/2 for atomoxetine were 1.9 ± 0.8 μg h/mL, 0.34 ± 0.11 μg/mL, 1.0 ± 0.5 h and 3.9 ± 1.3 h, respectively.     

Analysis of long-chain fatty acyl coenzyme a thioesters by negative ion fast-atom bombardment mass spectrometry and tandem mass spectrometry

Long-chain acyl Coenzyme A (CoA) is essentially composed of three major chemical groups, fatty acyl-, phosphopantetheino-, and 3′, 5′,-adenosine diphospho-moieties. The negative ion fast-atom bombardment mass spectrometry spectra of long-chain acyl CoA thioesters were characterized by the formation of abundant [M ? H]^? and two distinct classes of fragment ions, one class which retained the acyl group and another class which is related to CoA that contains the phosphopantethene and adenine. The ions which retained the acyl group in the spectrum of palmitoyl CoA appeared at m/z 675, 657, 595, and 577 and were found to decompose by loss of alkylketene observed at m/z 357 and 339. Those ions which retained the adenine group were observed at m/z 426 and 408. In contrast to these ions observed following fast-atom bombardment ionization, tandem mass spectrometry of the [M ? H]^?, from palmitoyl CoA (m/z 1004), yielded the adenine-containing ions as major products and the acyl-containing ions were of low abundance or not detected. These results suggested that the formation of many characteristic ions observed in direct FAB analysis occurred during the desorption process. The unique relationship between ions which involved the transition from acyl-containing ions to only CoA-containing ions by the loss of alkylketene allowed the development of tandem mass spectrometry protocols for the analysis of acyl CoA mixtures. Precursor scans of either m/z 357 or 339 yielded the identification of each species in a complex mixture. Identification of specific species was obtained with a neutral loss scan of the mass for a specific alkylketene.     

Biological Tissue Imaging with a Position and Time Sensitive Pixelated Detector

We demonstrate the capabilities of a highly parallel, active pixel detector for large-area, mass spectrometric imaging of biological tissue sections. A bare Timepix assembly (512?×?512 pixels) is combined with chevron microchannel plates on an ion microscope matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI TOF-MS). The detector assembly registers position- and time-resolved images of multiple m/z species in every measurement frame. We prove the applicability of the detection system to biomolecular mass spectrometry imaging on biologically relevant samples by mass-resolved images from Timepix measurements of a peptide?Cgrid benchmark sample and mouse testis tissue slices. Mass-spectral and localization information of analytes at physiologic concentrations are measured in MALDI-TOF-MS imaging experiments. We show a high spatial resolution (pixel size down to 740?×?740?nm² on the sample surface) and a spatial resolving power of 6???m with a microscope mode laser field of view of 100?C335???m. Automated, large-area imaging is demonstrated and the Timepix?? potential for fast, large-area image acquisition is highlighted.     

Simultaneous Quantification of Oroxylin A and Its Metabolite Oroxylin A-7-O-Glucuronide: Application to a Pharmacokinetic Study in Rat

A sensitive liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry (LC?CESI?CMS?CMS) method was developed and validated for the quantification of cepharanthine (CEP) in beagle dog plasma. The chromatographic separation was performed on an Agilent-C18 column and the mobile phase was composed of methanol:water with 10 mM ammonium acetate (20:80, v/v). Detection was operated in the positive ion mode and the tandem mass spectrometer was tuned in the multiple reactions monitoring mode (MRM) to monitor m/z transitions 607 ?? 365 for CEP and 285 ?? 193 for the internal standard (IS) diazepam. This method exhibited a linear range of 5?C2,500 ng mL^?1. The precision (RSD%) and accuracy (RME%) of the assay were <8.7 and 2.4%, respectively. The limit of quantification was 5 ng mL^?1 and no significant matrix effect was observed. The validated method has been successfully applied to pharmacokinetic study of CEP in beagle dog.     

Rapid and Sensitive LC−MS–MS Method for the Determination of Sarpogrelate in Human Plasma

A rapid and sensitive liquid chromatographic–tandem mass spectrometric method has been developed and validated for the estimation of sarpogrelate in human plasma. Sarpogrelate was extracted from human plasma by solid-phase extraction. Temocapril was used as the internal standard. Heated electron spray ionization mass spectrometry was performed on a TSQ Quantum Ultra MS system. The LC column was a Hypurity C₁₈ and the mobile phase was 2 mM ammonium formate (pH 3.00 ± 0.05):acetonitrile (30:70 v/v). A flow rate of 0.250 mL min^?1 was used. The quantitative analyses were carried out in the positive ion and full scan mode over the mass range m/z 60–500. The capillary, vaporiser temperatures were 325 and 200 °C respectively. The sheath gas pressure, spray voltage, collision energy and tube lense were 40, 3,500 V, 19 V, 198 V, respectively, and the mass spectra of the drugs were recorded by total ion monitoring. Retention times and characteristic mass fragments were recorded and the chosen diagnostic mass fragments were monitored in the mass chromatography mode. Signal intensities of each of the mass fragments: m/z 477 [M + H]⁺ for temocapril, m/z 430 [M + H]⁺ for sarpogrelate, were used for quantification. The calibration curves (the ratio between the peak areas as signal intensities of the drug analyzed and that of the internal standard (temocapril: m/z 477 [M + H]⁺) vs. the concentration of drug) exhibited linearity over the concentration range 5.00–2,500.00 ng mL^?1 human plasma. The recovery and the accuracy were calculated by comparing the peak areas as the signal intensities of each mass fragment for the drug in spiked samples after solid-phase extraction from human plasma to the peak area as the signal intensity of the mass fragment of internal standard sample. The method involves a rapid solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 5.0–2,500.0 ng mL^?1. The absolute recoveries for sarpogrelate (93.72%) and IS (91.42%) achieved from spiked plasma samples were consistent and reproducible.     

LC–Tandem-MS Development and Validation for the Determination of Tegaserod in Beagle Dog Plasma and Its Application to a Pharmacokinetic Study

A sensitive and selective liquid chromatography-tandem mass spectrometry method for quantitative determination of tegaserod was developed and validated over the linearity range 1.0–200.0 ng mL^?1 with 0.5 mL of plasma using diphenhydramine as an internal standard. Liquid–liquid extraction using ethyl ether was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring mode using the atmospheric pressure chemical ionization technique. The mobile phase consisted of methanol–water–formic acid (80:20:1, v/v/v), at a flow rate of 0.6 mL min^?1. In the positive mode, tegaserod produced a protonated precursor ion at m/z 302 and a corresponding product ion at m/z 173. The internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The intra- and inter-day accuracy at all levels fell in the ranges of 100.72–102.75% and 100.61–105.45%, and the intra- and inter-day precision were in the ranges of 4.20–5.74% and 1.90–4.17%, respectively. The method was successfully applied to a pharmacokinetic study of tegaserod after an oral administration of two kinds of tegaserod preparations to beagle dogs.     

LC�CMS�CMS Quantitative Determination of Brivudine in Human Plasma and Its Application to Pharmacokinetic Studies

A sensitive and specific liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry method has been developed and validated for the identification and quantification of brivudine in human plasma using diclofenac as an internal standard. The method involves extraction with ethyl acetate. The analyte was separated on a C₁₈ column and analyzed in multiple reaction monitoring mode with a negative electrospray ionization interface using the [M?CH]^? ions, m/z 332.8??m/z 80.9 for brivudine, m/z 293.6??m/z 249.5 for diclofenac. The method was validated over the concentration range of 5.54?C2,836 ??g L^?1 for brivudine. The intra-and inter-day precisions were less than 8.91% in terms of relative standard deviation (RSD), and the accuracy was within ?4.22% in terms of relative error (RE). The lower limit of quantification (LLOQ) was 5.54 ??g L^?1 with acceptable precision and accuracy. There were almost no matrix effects. Recovery of brivudine spiked in drug-free plasma was higher than 77.17%. The method was used to study the pharmacokinetic profile of brivudine in human plasma after oral administration of brivudine tablets.     

Rapid and Selective LC�CMS�CMS Method for the Determination of cyclo-trans-4-l-Hydroxyprolyl-l-serine (JBP485) in Rat Plasma

A rapid and selective liquid chromatographic/tandem mass spectrometric method for the determination of JBP485 was developed and validated. Following protein precipitation, the analyte and internal standard (JBP923) were separated from human plasma using an isocratic mobile phase on an Elite Kromasil C18 column. An API 3200 tandem mass spectrometer equipped with a Turbo ionSpray ionization source was used as the detector and operated in the positive ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 201.2 ?? 86.2 and m/z 219.2 ?? 86.2 was performed to quantify JBP485 and JBP923, respectively. The method was linear in the concentration range of 0.10?C50.00 ??g mL^?1 using 100 ??L of plasma. The lower limit of quantification was 0.10 ??g mL^?1. The intra- and inter-day relative standard deviations over the entire concentration range were less than 6.65%. Accuracy determined at three concentrations (0.25, 4.00 and 25.00 ??g mL^?1 for JBP485) ranged from ?0.78 to 2.74% in terms of relative error. Each plasma sample was chromatographed within 2.0 min. The method was successfully applied to characterize the pharmacokinetic profiles of JBP485 in rats after an intravenous injection of 6.25 mg kg^?1 JBP485.     

Simultaneous Determination of Pethidine and Atropine in Rabbit Plasma by LC�CMS�CMS and Its Application to a Pharmacokinetic Study after Intramuscular Administration

A sensitive and selective liquid chromatography?Ctandem mass spectrometry method for the determination of pethidine and atropine in rabbit plasma was developed and validated. The analytes and internal standard (IS) are extracted from plasma by liquid?Cliquid extraction using ethyl acetate, and separated on a Zorbax SB-Aq column (2.1 × 150 mm, 3.5 ??m) using acetonitrile?C0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode, and multiple reaction monitoring mode was used for quantification using target fragment ions m/z 247.8 ?? 219.7 for pethidine, m/z 289.9 ?? 123.8 for atropine and m/z 295.0 ?? 266.8 for IS, respectively. The assay is linear over the range of 5?C1,000 ng mL^?1 for pethidine and atropine, with a lower limit of quantification of 3 ng mL^?1 for pethidine and 5 ng mL^?1 for atropine. Intra-day and inter-day precision are less than 11% and the accuracy are in the range of 90.4?C106.3%. Furthermore, the newly developed method is successfully used for the determination of pethidine and atropine in rabbit plasma for pharmacokinetic study.