Rong-Hua Zhu

Determination of dansylated monoamine and amino acid neurotransmitters and their metabolites in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

The determination of neurotransmitters (NTs) and their metabolites facilitates better understanding of complex neurobiology in the central nervous system disorders and has expanding uses in many other fields. We present a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) method for the quantification of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), vanillymandelic acid (VMA), 3-methoxy-4-hydroxy phenylglycol (MHPG), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), glutamate (Glu), and gamma-aminobutyric acid (GABA). The NTs and their metabolites were dansylated and analyzed by an LC gradient on a C18 column on-line with a tandem mass spectrometer. This method exhibited excellent linearity for all of the analytes with regression coefficients higher than 0.99. The lower limit of quantification (LLOQ) values for DA, DOPAC, HVA, NE, VMA, MHPG, 5-HT, 5-HIAA, Glu, and GABA were 0.57, 0.37, 0.35, 0.40, 0.35, 0.91, 0.27, 0.43, 0.65, and 1.62 pmol/ml, respectively. The precision results were expressed as coefficients of variation (CVs), ranging from 1.5% to 13.6% for intraassay and from 2.9% to 13.7% for the interassay. This novel LC-ESI/MS/MS approach is precise, highly sensitive, specific, and sufficiently simple. It can provide an alternative method for the quantification of the NTs and their metabolites in human plasma.

Simultaneous determination of isoniazid, rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry

A rapid and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250mmx4.6mm, 5.0microm, USA) at 25 degrees C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11-5.42microg/g in tissues and 0.18-9.04microg/ml in plasma, for RFP were 0.12-1200microg/g in tissues and 4.0-200microg/ml in plasma, and for LVX were 0.13-26.2microg/g in tissues and 0.09-4.53microg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13microg/g and for those in mouse plasma were 18.1, 20.0 and 21.8ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05microg/g and for those in mouse plasma were 5.5, 6.0 and 6.6ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.

MultiSimplex optimization of chromatographic separation and dansyl derivatization conditions in the ultra performance liquid chromatography-tandem mass spectrometry analysis of risperidone, 9-hydroxyrisperidone, monoamine and amino acid neurotransmitters in human urine.

A pre-column dansylated ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of risperidone (RIP), 9-hydroxyrisperidone (9-OH-RIP), monoamine and amino acid neurotransmitters in human urine was developed with the aim of providing data on how neurotransmitters may influence each other or change simultaneously in response to risperidone treatment. MultiSimplex based on the simplex algorithm and the fuzzy set theory was applied to the optimization of chromatographic separation and dansyl derivatization conditions during method development. This method exhibited excellent linearity for all the analytes with regression coefficients higher than 0.997. The lower limit of quantification (LLOQ) values for 9-OH-RIP and RIP were 0.11 and 0.06 ng/ml, respectively, and for neurotrasmitters ranged from 0.31 to 12.8 nM. The mean accuracy ranged from 94.7% to 108.5%. The mean recovery varied between 81.6% and 97.5%. All the RSD of precision and stability were below 9.7%. Finally, the optimized method was applied to analyze the first morning urine samples of schizophrenic patients treated with risperidone and healthy volunteers.

Validated LC-MS (ESI) assay for the simultaneous determination of amitriptyline and its metabolite nortriptyline in rat plasma: Application to a pharmacokinetic comparison

A rapid, sensitive and specific method based on high performance liquid chromatography with electrospray ionization mass spectrometry (HPLC-MS/ESI) has been developed for the simultaneous determination of amitriptyline and nortriptyline in rat plasma. Sample preparation involved liquid-liquid extraction with methyl t-butyl ether after alkalified with 0.5 mol/l NaOH. Chromatographic separation was performed on a XB-C4 column (4.6 mm x 250 mm, 5 microm, Welch Materials) with a mobile phase consisting of 10mM ammonium acetate (0.6 per thousand formic acid)-acetonitrile (60:40, v/v) at a flow rate of 1.0 ml/min. Calibration curves were linear within the ranges of 10-3200 ng/ml for amitriptyline and 10-1000 ng/ml for nortriptyline. This method was successfully applied to the pharmacokinetic study in rats after intravenous injection of amitriptyline hydrochloride.

Multiple dose pharmacokinetics of risperidone and 9-hydroxyrisperidone in Chinese female patients with schizophrenia

AbstractAim:To study the multiple dose clinical pharmacokinetics of risperidone and its main active metabolite, 9-hydroxyrisperidone, in Chinese female patients with schizophrenia.Methods:The subjects were 23 Chinese female inpatients aged 18–65 years who met the CCMD-III (third revision of the Chinese Criteria of Mental Disorders) criteria for schizophrenia. Subjects were tested after 17 d of treatment with 2 mg risperidone twice daily. Plasma concentrations of risperidone and 9-hydroxy-risperidone were assayed by using validated high performance liquid chromatography-mass spectrometry (HPLC-MS) methods.Results:Risperidone was rapidly absorbed (Tmax was 1.6 h) and its T1/2 in plasma was short (3.2 h). 9-hydroxy-risperidone was quickly metabolized from the parent drug with a mean Tmax of 2.5 h. It had a long half-life of 24.7 h. The Cssav of risperidone and 9-hydroxyrisperidone were 36.9±33.1 and 110.6±30.5 μg·h·L−1, respectively, and the AUCss0–12 were 443.2±397.4 and 1327.2±402.3 μg·h·L−1, respectively. CL/F and V/F of risperidone were 8.7±6.2 L/h and 34.1±24.3 L, respectively. Interindividual variations for pharmacokinetic parameters were quite large for risperidone. All 23 subjects experienced high prolactin levels when treated with risperidone. However there was no correlation between prolactin level and the concentration of risperidone, 9-hydroxy-risperidone, or the active moiety.Conclusion:Risperidone showed large interindividual variations in pharmacokinetics. Administration of risperidone resulted in high serum prolactin levels. The results indicate that systemic exposure to risperidone and 9-hydroxy-risperidone in female Chinese schizophrenic patients is higher relative to published data for white Caucasian patients. Larger studies regarding the PK/PD relationship may be required to develop a reasonable clinical dosage regimen for Chinese female patients.

Ultra-performance liquid chromatography–tandem mass spectrometric method for the determination of strychnine and brucine in mice plasma

A selective, simple and efficient method-ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for determination of two toxic alkaloids, namely strychnine and brucine in mice plasma. The UPLC separation was carried out using a 1.7 μm BEH C(18) column (50 mm × 2.1 mm) with a mobile phase consisting of methanol:0.1% formic acid (25:75, v/v), hence providing high efficiency, high resolution and excellent peak shape for the analytes and internal standard. The method was validated over the range of 2.48-496.4 ng/ml for strychnine and 2.64-528 ng/ml for brucine, respectively. Intra- and inter-day accuracy ranged from 95.0% to 107.9% for strychnine, 93.4% to 103.3% for brucine, and the precisions were within 13.8%. The extraction recoveries of both the two alkaloids exceed 81.9%. With a simple and minor sample preparation procedure and short run-time (<3 min), the proposed method was applicable for the pharmacokinetic and toxicological analysis of strychnine and brucine in vivo.

Simultaneous quantitative determination of paracetamol and its glucuronide conjugate in human plasma and urine by liquid chromatography coupled to electrospray tandem mass spectrometry: application to a clinical pharmacokinetic study.

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of paracetamol (APAP) and its glucuronide conjugate (PG) in human plasma and urine. Plasma samples were precipitated with the mixture of acetonitrile and propylene glycol (90:10, v/v) solution and urine samples were diluted with the mobile phase, which were used to isolate the analytes from biological matrices followed by injection of the extracts onto a C(18) column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI(+)). The method was validated over the concentration range of 10-30,000 ng/mL and 100-6000 ng/mL for APAP in human plasma and urine as well as 10-15,000 ng/mL and 200-60,000 ng/mL for PG in human plasma and urine, respectively. Inter- and intra-run precisions of APAP and PG were less than 15% and the accuracy was within 85-115% for both plasma and urine. The average extraction recoveries were 93.1% and 89.1% for APAP, and 93.7% and 92.3% for PG in human plasma and urine, respectively. The linearity, recovery and stability were validated for APAP and PG in human plasma and urine. The method proved to be simple, robust and time efficient.

Validated LC‐MS/MS assay for the quantitative determination of nalbuphine in human plasma and its application to a pharmacokinetic study

A solid-phase extraction-liquid chromatographic-tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™-X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 μm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 → 340 for nalbuphine and m/z 328 → 310 for naloxone. The assay was linear over the concentration range 0.50-500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal-to-noise ratio of 44.79. The intra- and inter-day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine.

The effects of food on the pharmacokinetics of mitiglinide tablets in healthy volunteers and a novel mass-spectrometric (UPLC-MS/MS) method for such studies

What is known and Objective:  Mitiglinide (MGN) is a new insulinotropic agent of the glinide class with rapid onset. The effects of food intake on the pharmacokinetic (PK) profile of mitiglinide tablets after single oral administration have not yet been reported in healthy adults. We aimed to assess the effects of food intake on the PK properties of mitiglinide (MGN) tablets, using a novel analytical method, after single escalating oral doses in healthy Chinese volunteers.

Quantitative analysis of erythromycylamine in human plasma by liquid chromatography-tandem mass spectrometry and its application in a bioequivalence study of dirithromycin enteric-coated tablets with a special focus on the fragmentation pattern and carryover effect

A liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of erythromycylamine, which is the predominant active metabolite of dirithromycin in human plasma. After solid-phase extraction, the analyte and internal standard (IS) were separated by using an isocratic mobile phase consisting of 20 mM ammonium acetate (pH 3.9, adjusted with formic acid)-acetonitrile (75:25, v/v) on a Phenyl-Hexyl column (150 × 2.1 mm, 3 μm) and then analyzed in positive ion mode under electrospray ionization. Azithromycin was selected as the IS because it has the most similar mass spectrometric and chromatographic behaviors to the analyte. The respective multiple reaction monitoring (MRM) transitions, m/z 368.5>83.2 for erythromycylamine and m/z 375.4>115.2 for IS were chosen to achieve high sensitivity and selectivity in determination. A more acidic mobile phase (pH 3.9) than those of previous reports and a special needle wash (ethylene glycol-acetonitrile-water, 50:30:20, v/v/v, adjusted to pH 3.9 using formic acid) were used to eliminate the carryover effects of the two macrolides. The method exhibited a linear dynamic range of 0.5-440.0 ng/mL for erythromycylamine in human plasma (r=0.9999). The lower limit of quantification (LLOQ) and limit of detection (LOD) were 0.5 and 0.05 ng/mL, respectively. The mean extraction recoveries were higher than 94.0% for the analyte and IS. The intra- and inter-day precisions ranged from 1.4 to 5.4% and from 1.6 to 4.0%, respectively. The accuracy varied between 91.2 and 101.2%. The established method was successfully applied to analyze the human plasma samples from 24 healthy subjects in a bioequivalence study of two dirithromycin enteric-coated formulations.