Kinetex C18 Column Research Articles

Multicriteria Optimization Methodology in Stability-Indicating Method Development of Cilazapril and Hydrochlorothiazide.

Multicriteria optimization methodology was applied in development of UHPLC-UV-MS method for separation of cilazapril, hydrochlorothiazide and their degradation products. This method is also applicable for analysis of cilazapril, hydrochlorothiazide and their degradation products in combined tablet formulation. Prior to method optimization forced degradation studies were conducted. Cilazapril and hydrochlorothiazide were subjected to acidic (0.1, 0.5 and 1.0 M HCl), basic (0.1, 0.5 and 1.0 M NaOH), thermal (70°C), oxidative (3-30% H2O2) degradation and photodegradation (day light). Cilazapril appeared to be unstable toward acid and base and resulted in formation of cilazaprilat. Hydrochlorothiazide significantly degraded after acid, base and thermal hydrolysis and formed degradation product was 4-amino-6-chlorobenzene-1.3-disulfonamide. For both substances, after oxidative degradation unknown products have arisen. Initial percentage of acetonitrile in mobile phase, final percentage of acetonitrile in mobile phase, time of gradient elution and column temperature were defined as variables to be optimized toward two chromatographic responses by means of central composite design and Derringer's desirability function. The satisfactory chromatographic analysis was achieved on Kinetex C18 (2.6 µm, 50 × 2.1 mm) column with temperature set at 25°C. The final mobile phase consisted of acetonitrile and 20 mM ammonium formate buffer (pH adjusted to 8.5). The flow rate of the mobile phase was400 μL min-1 and it was pumped in a gradient elution mode.

A novel LC–MS/MS method for the simultaneous quantification of topiramate and its main metabolites in human plasma

The aim of the present report was to develop and validate simple, sensitive and reliable LC–MS/MS method for quantification of topiramate (TPM) and its main metabolites: 2,3-desisopropylidene TPM, 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM in human plasma samples. The most abundant metabolite 2,3-desisopropylidene TPM was isolated from patients urine, characterized and afterwards used as an authentic standard for method development and validation. Sample preparation method employs 100μL of plasma sample and liquid–liquid extraction with a mixture of ethyl acetate and diethyl ether as extraction solvent. Chromatographic separation was achieved on a 1290 Infinity UHPLC coupled to 6460 Triple Quad Mass Spectrometer operated in negative MRM mode using Kinetex C18 column (50×2.1mm, 2.6μm) by gradient elution using water and methanol as a mobile phase and stable isotope labeled TPM as internal standard. The method showed to be selective, accurate, precise and linear over the concentration ranges of 0.10–20μg/mL for TPM, 0.01–2.0μg/mL for 2,3-desisopropylidene TPM, and 0.001–0.200μg/mL for 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM. The described method is the first fully validated method capable of simultaneous determination of TPM and its main metabolites in plasma over the selected analytical range. The suitability of the method was successfully demonstrated by the quantification of all analytes in plasma samples of patients with epilepsy and can be considered as reliable analytical tool for future investigations of the TPM metabolism.

Determination of meropenem levels in human serum by high-performance liquid chromatography with ultraviolet detection.

Meropenem is a β-lactam broad-spectrum antibiotic and belongs to the subgroup of carbapenems. It is primarily used in intensive care units for intravenous treatment of severe infections. To avoid bacterial resistance or toxic side effects, the determination of serum meropenem concentration is highly advisable. A simple and fast method for the quantitative determination of meropenem in human serum using high-performance liquid chromatography with ultraviolet detection (HPLC/UV) was developed and validated. Meropenem was determined by an isocratic HPLC using a tris(hydroxymethyl)aminomethane buffer (pH 8.5; 15% methanol) as a mobile phase and UV detection at 300 nm, with a flow rate of 1.0 mL/min and an analysis time of 10 min. Chromatographic separation was performed on a Kinetex C18 column (5 μm, 150 × 4.6 mm). In order to remove undesired serum components, solid-phase extraction was used for sample preparation. Since meropenem is not stable in solution, sample and stock solution were stored at -80°C. After preparation, samples were stable at room temperature for at least 6 h. The calibration curve was linear from 3.5 to 200 mg/L with a correlation coefficient r2 of 0.999. The method is accurate with an intra- and inter-assay precision <18.5%.

LC–MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and DM1 as potential intracellular catabolites of the antibody-drug conjugate trastuzumab emtansine (T-DM1)

Lysine-MCC-DM1, MCC-DM1 and DM1 are potential catabolites of trastuzumab emtansine (T-DM1). A convenient liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated to detect these catabolites simultaneously in in vitro investigations for the first time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100×2.1mm, 2.6μm) with mobile-phase gradient elution. The calibration curves of each analyte ranging from 1 to 100nM showed good linearity (r2>0.995). The method was validated successfully and applied to the intracellular catabolism and regulation of T-DM1.

Development of Solid-Phase Extraction and HPLC Method for Simultaneous Estimation of Ilaprazole and Glimepiride in Rat Plasma: Application to Pharmacokinetic Studies.

A novel, simple and mass spectrometry (MS) compatible high-performance liquid chromatography (HPLC) method is reported for the simultaneous estimation of ilaprazole (ILA) and glimepiride (GLM) in rat plasma. The bio-analytical procedure involves extraction of ILA, GLM and internal standard (IS) from rat plasma with a solid-phase extraction (SPE) process. The chromatographic analysis was performed on Waters-600 system using an isocratic mobile phase comprising methanol:water (80:20 % v/v) with pH of water modified to three using formic acid at a flow rate of 1.0 mL/min and Kinetex C18 column maintained at 30 ± 1°C. The signals were monitored using a PDA detector set at 225 nm. IS, ILA and GLM eluted at 2.04, 4.7 and 7.4 min, respectively, and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 10-600 ng/mL (r2 = 0.999). The intra- and inter-day precisions for ILA and GLM were (%RSD values) in the range of 1.52-9.74 and 1.52-11.76%, respectively, in rat plasma. The method was successfully applied in pharmacokinetic studies followed by oral administration of GLM and ILA in rats.

Absolute oral bioavailability of fenofibric acid and choline fenofibrate in rats determined by ultra-performance liquid chromatography tandem mass spectrometry.

Choline fenofibrate is the choline salt of fenofibric acid, which releases free fenofibric acid in the gastrointestinal tract. To estimate the absolute oral bioavailability of fenofibric acid and choline fenofibrate, a novel and sensitive UPLC-MS/MS method with liquid-liquid extraction procedure was developed for the determination of fenofibric acid in rat plasma. The separation was achieved on a Phenomenex Kinetex C18 column (50 × 2.1 mm, 2.6 μm) containing 2 mm ammonium acetate-methanol with a gradient elution program. Validations of this method including specificity, sensitivity (limit of quantification, 5 ng/mL), linearity (0.005-10 μg/mL), accuracy (within ±4.3%), precision (intra- and inter-day coefficient of variation <11.3%), recovery (94.9-105.2% for fenofibric acid), matrix effect, stability and dilution, were all within acceptable limits. This method successfully supported the determination of fenofibric acid and choline fenofibrate. The absolute oral bioavailability was 93.4% for choline fenofibrate and 40.0% for fenofibric acid. These results suggested that choline fenofibrate and fenofibric acid had a better in vivo pharmacokinetic behavior than that of fenofibrate. The two new orally administrated pharmaceuticals, fenofibric acid and choline fenofibrate, can be developed as alternatives to fenofibrate.

Simultaneous determination of β-sitosterol, campesterol, and stigmasterol in rat plasma by using LC-APCI-MS/MS: Application in a pharmacokinetic study of a titrated extract of the unsaponifiable fraction of Zea mays L.

A liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated to investigate the pharmacokinetic properties of β-sitosterol, campesterol, and stigmasterol in rat plasma. Cholesterol-d6 was used as an internal standard. To avoid interference of the three phytosterols in rat plasma and minimize matrix effects, a small volume (10 μL) of 4% bovine serum albumin was used as a surrogate matrix for making calibrators and quality control samples. Rat plasma (10 μL) samples were extracted by liquid-liquid extraction with methyl tert-butyl ether and separated on a Kinetex C18 column. The detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode using positive atmospheric pressure chemical ionization. This assay was linear over concentration ranges of 250-5000 ng/mL (β-sitosterol), 250-5000 ng/mL (campesterol), and 50-2000 ng/mL (stigmasterol). Additionally, a second set of quality controls made in rat plasma was also evaluated against calibration curves made using the surrogate matrix. All the validation data, including the specificity, precision, accuracy, recovery, matrix effect, stability, and incurred sample reanalysis conformed to the acceptance requirements. Our method was successfully applied to study the pharmacokinetics of three phytosterols in rats.

A validated, sensitive HPLC-MS/MS method for quantification of cis-para-methyl-4-methylaminorex (cis-4,4'-DMAR) in rat and human plasma: application to pharmacokinetic studies in rats.

4,4'-DMAR is an analogue of the known psychostimulants 4-methylaminorex and aminorex. In the light of reports of deaths associated with its abuse, and the easy access from Internet vendors, the EU Council recently decided on control measures across member states. Here we describe a validated method for measuring plasma levels of cis-4,4'-DMAR, crucial for preclinical studies and analysis in human plasma. Chromatographic separation was done by gradient elution on a Kinetex C18 column with 0.1% formic acid in water and 0.1% formic acid in acetonitrile at 0.2 mL/min. Detection was by positive electrospray ionization (ESI+) in multiple reaction monitoring mode monitoring the quantifier transitions m/z 191.4 → m/z 148.3 for cis-4,4'-DMAR and m/z 259.3 → m/z 194.2 for carbamazepine (internal standard). Protein precipitation with 1% of formic acid in acetonitrile was used in cis-4,4'-DMAR extraction from plasma; recovery was high (>93%) with a negligible matrix effect. This method provides an accurate, precise, and sensitive method for cis-4,4'-DMAR quantification in human and rat plasma, following European Medicine Agency guidelines for bioanalytical method validation. Pharmacokinetic studies were conducted in rats. After an intravenous dose of 1 mg/kg, plasma levels declined rapidly (≥80% in 4 h), followed by a slow elimination phase (t1/2 of 5.14 ± 0.65 h). Absorption was rapid after intraperitoneal injection (tmax = 15 min) with a rapid decline thereafter; Cmax and AUC0-240min showed dose-proportionality over the dose range 1-10 mg/kg. This method was successfully applied to investigate pharmacokinetic properties in rats and could be used to quantify cis-4,4'-DMAR levels in human plasma. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a UPLC–MS/MS method for the determination of lomefloxacin in rabbit aqueous humor and its application to a pharmacokinetic study

Lomefloxacin is a kind of synthetic fluoroquinolone antibiotic, which is used for the treatment of infectious diseases. In this study, a rapid and efficient liquid chromatography-tandem mass spectrometric assay was developed to determine the concentration of lomefloxacin in rabbit aqueous humor quantitatively. Aqueous humor samples were extracted by protein precipitation. Ofloxacin was chosen as internal standard. The chromatographic separation was achieved on a Kinetex C18 (50mm×2.10mm, 2.6μm, Phenomenex Corp, USA) column, with a gradient of methanol (0.1% formic acid) and water (0.1% formic acid). Multiple reaction monitoring (MRM) with positive ionization mode was used for the mass analysis. The validation of this method was based on the European Medicines Agency (2011) [1] and US FDA Guidelines (2001) [2]. The calibration range of aqueous humor samples was 5-1200ng/mL with r=0.9990 (n=6). For all QC samples, Inter-and intra-run precisions were less than 15% and accuracies were between 80%-120%. In conclusion, the assay was rapid, sensitive and able to determinate the lomefloxacin in rabbit aqueous humor accurately. At the same time, this method was successfully applied to study the pharmacokinetics of lomefloxacin hydrochloride eye drops and lomefloxacin hydrochloride ophthalmic gel in rabbit aqueous humor.

Assessment of pharmacokinetics, bioavailability and protein binding of anacetrapib in rats by a simple high‐performance liquid chromatography–tandem mass spectrometry method

Anacetrapib is a potent and selective CETP inhibitor and is undergoing phase III clinical trials for the treatment of dyslipidemia. A simple and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the quantification of anacetrapib in rat plasma was developed and validated using an easily purchasable compound, chlorpropamide, as an internal standard (IS). A minimal volume of rat plasma sample (20 μL) was prepared by a single-step deproteinization procedure with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C18 column with a gradient mobile phase consisting of water and acetonitrile containing 0.1% formic acid at a flow rate of 0.3 mL/min. Mass spectrometric detection was performed using selected reaction monitoring modes at the mass/charge transitions m/z 638 → 283 for anacetrapib and m/z 277 → 175 for IS. The assay was validated to demonstrate the selectivity, linearity, precision, accuracy, recovery, matrix effect and stability. The lower limit of quantification was 5 ng/mL. This LC-MS/MS assay was successfully applied in the rat plasma protein binding and pharmacokinetic studies of anacetrapib. The fraction of unbound anacetrapib was determined to be low (ranging from 5.66 to 12.3%), and the absolute oral bioavailability of anacetrapib was 32.7%.

Simultaneous quantification of 11 cannabinoids and metabolites in human urine by liquid chromatography tandem mass spectrometry using WAX-S tips.

A comprehensive cannabinoid urine quantification method may improve clinical and forensic result interpretation and is necessary to support our clinical research. A liquid chromatography tandem mass spectrometry quantification method for ∆(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), ∆(9)-tetrahydrocannabinolic acid (THCAA), cannabinol (CBN), cannabidiol (CBD), cannabigerol (CBG), ∆(9)-tetrahydrocannabivarin (THCV), 11-nor-9-carboxy-THCV (THCVCOOH), THC-glucuronide (THC-gluc), and THCCOOH-glucuronide (THCCOOH-gluc) in urine was developed and validated according to the Scientific Working Group on Toxicology guidelines. Sample preparation consisted of disposable pipette extraction (WAX-S) of 200μL urine. Separation was achieved on a Kinetex C18 column using gradient elution with flow rate 0.5mL/min, mobile phase A (10mM ammonium acetate in water), and mobile phase B (15% methanol in acetonitrile). Total run time was 14min. Analytes were monitored in both positive and negative ionization modes by scheduled multiple reaction monitoring. Linear ranges were 0.5-100μg/L for THC and THCCOOH; 0.5-50μg/L for 11-OH-THC, CBD, CBN, THCAA, and THC-gluc; 1-100μg/L for CBG, THCV, and THCVCOOH; and 5-500μg/L for THCCOOH-gluc (R (2) > 0.99). Analytical biases were 88.3-113.7%, imprecisions 3.3-14.3%, extraction efficiencies 42.4-81.5%, and matrix effect -10 to 32.5%. We developed and validated a comprehensive, simple, and rapid LC-MS/MS cannabinoid urine method for quantification of 11 cannabinoids and metabolites. This method is being used in a controlled cannabis administration study, investigating urine cannabinoid markers documenting recent cannabis use, chronic frequent smoking, or route of drug administration and potentially improving urine cannabinoid result interpretation.

Simultaneous determination of parecoxib sodium and its active metabolite valdecoxib in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study after intravenous and intramuscular administration.

In this study, we developed and validated a new, rapid, specific and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to simultaneously determine parecoxib sodium (PX) and its active metabolite, valdecoxib (VX), in rat plasma. Plasma samples were prepared by plasma protein precipitation combined with a liquid-liquid extraction method. The separation was carried out on a Kinetex C18 column (2.1mm×50mm, 2.6μm) with a gradient elution using methanol (A) and a 2mM ammonium acetate aqueous solution (B). The analysis was performed in less than 3min with a flow rate of 0.2mL/min. Ketoprofen was used as an internal standard (IS). Mass spectrometric detection was conducted with a triple quadrupole detector equipped with electrospray ionization in the negative ion mode (ESI(-)) using multiple reaction monitoring (MRM). The calibration curves were linear over the concentration ranges of 5-4000ng/mL for PX and 5-2000ng/mL for VX with all correlation coefficients greater than 0.998. The intra- and inter-day relative standard deviations (RSD) for both analytes were within 15% and the accuracy was within 85-115% at all quality control levels. The mean extraction recoveries for all analytes obtained from three concentrations of QC plasma samples were more than 89.0% efficient. Selectivity, matrix effect, dilution integrity and stability were also validated. The method was successfully used to investigate the pharmacokinetics of PX and VX in rat plasma after intravenous and intramuscular administration of PX.

Simultaneous analysis of seven marker compounds from Saposhnikoviae Radix, Glehniae Radix and Peucedani Japonici Radix by HPLC/PDA.

A new combination of high performance liquid chromatography (HPLC) method coupled with photodiode array (PDA) analysis has been developed for the simultaneous quantitative determination of seven major components in Saposhnikoviae Radix (SR), Glehniae Radix (GR) and Peucedani Japonici Radix (PR), namely peucedanol 7-O-β-D-glucopyranoside (1), prim-O-glucosylcimifugin (2), cimifugin (3), 4'-O-β-D-glucosyl-5-O-methylvisamminol (4), bergapten (5), sec-O-glucosylhamaudol (6), and imperatorin (7). Clear separation of these seven components were achieved on a Phenomenex Kinetex C18 (250×4.6mm, 5μm) column by gradient elution of water (A) and methanol (B) as mobile phase. The flow rate was 1.0mL/min and the UV detector wavelength was set at 254nm. The method was successfully used in the analysis of SR, GR, and PR with relatively simple conditions and procedures, and the results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The results indicate that the established HPLC/PDA method is suitable for the classification of SR, GR, and PR.

Quantitative Determination of Spermidine in 50 German Cheese Samples on a Core-Shell Column by High-Performance Liquid Chromatography with a Photodiode Array Detector Using a Fully Validated Method.

In the current study, the spermidine (8) contents of 51 German and 9 international cheese samples (from France, Ireland, Italy, The Netherlands, and Switzerland) were analyzed by a modified and fully validated method using high-performance liquid chromatography with photodiode array detection. After precolumn derivatization of biogenic amines with dansyl chloride (11), the compounds were separated on a Kinetex C18 column and detected at λ = 254 nm. This method for compound 8 analysis in cheese was validated for the first time according to U.S. Food and Drug Administration (FDA) guidelines for bioanalytical method validation with regard to selectivity, precision, accuracy, recovery, linearity, lower limit of detection (LOD), lower limit of quantitation (LOQ), standard solution stability, short- and long-term stability, freeze-thaw stability, and benchtop stability. The detector response was linear from 0.002 to 8 mg/L 8 (R(2) > 0.999). Low LOD and LOQ values of 1 and 2 μg/L, respectively, reflected the high sensitivity of the method. The intra- and interday recoveries of the 8-spiked cheese samples ranged between 87.7 and 102.6%. This validated method was selective, accurate, and precise and was successfully applied for the quantitative analysis of compound 8 in 60 cheese samples. Furthermore, the simultaneous detection of eight additional biogenic amines is possible but not validated.

Determination of 6258-70, a new semi-synthetic taxane, in rat plasma and tissues: Application to the pharmacokinetics and tissue distribution study

Cancer is the leading cause of death all over the world. Among the chemotherapy drugs, taxanes play an important role in cancer treatment. 6258-70 is a new semi-synthetic taxane which has a broad spectrum of antitumor activity. A fast and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed for quantification of 6258-70 in rat plasma and tissues in this paper. After extraction by liquid-liquid extraction method with methyl tert-butyl ether, the samples were separated on a Kinetex C18 column (50mm×2.1mm, 2.6µm, Phenomenex, USA) within 3min. The method was fully validated with the matrix effect between 87.7% and 99.5% and the recovery ranging from 80.3% to 90.1%. The intra- and inter-day precisions were less than 9.5% and the accuracy ranged from −3.8% to 6.5%. The reliable method was successfully applied to the pharmacokinetics and tissue distribution studies of 6258-70 after intravenous administration in rats. The pharmacokinetic results indicated that the pharmacokinetic behavior of 6258-70 in rats was in accordance with linear features within tested dosage of 1 to 4mg/kg, and there was no significant difference between the two genders. The tissue distribution study showed that 6258-70 had an effective penetration, spread widely and rapidly and could cross blood-brain barrier. The results of pharmacokinetics and tissue distribution may provide a guide for future study.

Optimization of Microwave-Assisted Extraction Conditions for Five Major Bioactive Compounds from Flos Sophorae Immaturus (Cultivars of Sophora japonica L.) Using Response Surface Methodology.

Microwave-assisted extraction was applied to extract rutin; quercetin; genistein; kaempferol; and isorhamnetin from Flos Sophorae Immaturus. Six independent variables; namely; solvent type; particle size; extraction frequency; liquid-to-solid ratio; microwave power; and extraction time were examined. Response surface methodology using a central composite design was employed to optimize experimental conditions (liquid-to-solid ratio; microwave power; and extraction time) based on the results of single factor tests to extract the five major components in Flos Sophorae Immaturus. Experimental data were fitted to a second-order polynomial equation using multiple regression analysis. Data were also analyzed using appropriate statistical methods. Optimal extraction conditions were as follows: extraction solvent; 100% methanol; particle size; 100 mesh; extraction frequency; 1; liquid-to-solid ratio; 50:1; microwave power; 287 W; and extraction time; 80 s. A rapid and sensitive ultra-high performance liquid chromatography method coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (EIS-Q-TOF MS/MS) was developed and validated for the simultaneous determination of rutin; quercetin; genistein; kaempferol; and isorhamnetin in Flos Sophorae Immaturus. Chromatographic separation was accomplished on a Kinetex C18 column (100 mm × 2.1 mm; 2.6 μm) at 40 °C within 5 min. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile (71:29; v/v). Isocratic elution was carried out at a flow rate of 0.35 mL/min. The constituents of Flos Sophorae Immaturus were simultaneously identified by EIS-Q-TOF MS/MS in multiple reaction monitoring mode. During quantitative analysis; all of the calibration curves showed good linear relationships (R2 > 0.999) within the tested ranges; and mean recoveries ranged from 96.0216% to 101.0601%. The precision determined through intra- and inter-day studies showed an RSD% of <2.833%. These results demonstrate that the developed method is accurate and effective and could be readily utilized for the comprehensive quality control of Flos Sophorae Immaturus.

Simultaneous determination of seven alkaloids in rat plasma by UFLC-MS/MS and its application to a pharmacokinetic study after oral administration of Cerebralcare Granule.

An ultra fast liquid chromatography-tandem mass sepectrometry (UFLC-MS/MS) method was developed for simultaneous determination of seven active alkaloid components (tetrahydropalmatine, corydaline, α-allocryptopine, tetrahydroberberine, tetrahydrocoptisine, tetrahydrocolumbamine and dehydrocorydaline) in rat plasma after oral administration of Cerebralcare Granule. Plasma samples were pretreated by protein precipitation with acetronitrile containing the internal standard diazepam. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100×2.1mm, 2.6μm) with gradient elution using mobile phase consisting of acetonitrile -0.1% formic acid in water at a flow rate of 0.3mL/min. The detection was performed on an electrospray ionization triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM) with positive ionization mode. The established method was fully validated and proved to be sensitive and specific with lower limits of quantification (LLOQs) all less than 0.0265ng/mL in rat plasma. Good linearities of seven alkaloids were obtained in respective concentration ranges (r>0.9923). The intra- and inter-day precisions were below of 15% for all the seven alkaloids in terms of relative standard deviation (RSD), and the accuracies were ranged from -2.7% to 8.3% in terms of relative error (RE). Extraction recovery, matrix effect and stability were within the required limits in rat plasma. The validated method was successfully applied to investigate the pharmacokinetics of the seven alkaloids in rat plasma after oral administration of Cerebralcare Granule (CG).

Accurate Method of HPLC-Ms/Ms Determination of Mycophenolic Acid in Human Plasma

A simple, accurate and precise method was developed using HPLC-MS/MS to quantify mycophenolic acid in human plasma. Mycophenolic acid-D3 was used as internal standard. Sample preparation involved protein precipitation. Chromatographic analysis was performed on a Phenomenex Kinetex C18 (30 mm × 4.6 mm, 2.6 μm) column using acetonitrile-water as the mobile phase. Detection in negative ionization mode was used to quantify mycophenolic acid by multiple reaction monitoring. The calibration curve was linear in the range of 0.5-30 μg/mL, with intra-and inter-batch values for accuracy and precision (coefficient of variation) ranging from 99.76 to 111.38% and from 2.54 to 9.01%, respectively. The method was successfully applied to conduct a pharmacokinetic study of 360 mg coated tablet Myfortic in 48 healthy participants; 768 blood samples were analysed.

High-Throughput Quantification of Monofluoroacetate (1080) in Milk as a Response to an Extortion Threat

As a food defense measure against an extortion threat to poison infant formula with monofluoroacetate, a robust methodology for monofluoroacetate analysis in fluid milk and powdered dairy products was developed and optimized. Critical challenges posed by this situation required that the analytical methodology provide (i) high specificity, (ii) high throughput capable of analyzing thousands of samples of fluid milk per day, and (iii) trace-level detection of 1 ng/g or lower to achieve the maximum residue limit. Solid-phase extraction-purified acetone extracts of fluid milk were derivatized with aniline, and after ultrahigh-performance liquid chromatography using a Kinetex-C18 column packed with 1.3-μm shell particles, the resulting N-phenyl 2-fluoroacetamide could be determined by liquid chromatography-tandem mass spectrometry in a highly specific manner and with a limit of quantification of 0.5 ng/ml. By using 4-(4-chlorophenoxy)aniline as a derivatizing agent, the method could be extended to powdered dairy products with the same limit of quantification. Between January and July 2015, some 136,000 fluid milk samples were tested using this method. This analytical testing of fluid milk formed one element in a larger program of work by multiple agencies to ensure that consumers could continue to have confidence in the safety of New Zealand milk and dairy products.

LC–MS/MS method development for quantification of busulfan in human plasma and its application in pharmacokinetic study

A simple, rapid, specific and precise liquid chromatography—tandem mass spectrophotometric (LC–MS/MS) method was developed and validated for quantification of busulfan, in human plasma. busulfan d8 was used as internal standard, added to plasma sample prior to extraction using acetonitrile as a precipitating agent. Chromatographic separation was achieved on phenomenex kinetex C18 column (50mm×2.1mm, 2.6μm) with acteonitrile: 10mM ammonium formate buffer (80:20v/v) as an isocratic mobile phase with a flow rate of 0.5mLmin−1. Quantitation was performed by transition of 264.1→151.1 (m/z) for busulfan and 272.1→159.1 (m/z) for busulfan d8. The lower limit of quantitation was 0.2ngmL−1 with a 100μL plasma sample. The concentrations of nine working standards showed linearity between 0.2 and 100ngmL−1 (r2≥0.9986). Chromatographic separation was achieved within 2.0min. The average extraction recoveries of 3quality control concentrations were 92.52% for busulfan and 90.75% for busulfan d8. The coefficient of variation was ≤15% for intra- and inter-batch assays. The developed method was successfully applied for the determination of Busulfan pharmacokinetics after oral administration.