Sanwang Li

New techniques of on-line biological sample processing and their application in the field of biopharmaceutical analysis

Biological sample pretreatment is an important step in biological sample analysis. Due to the diversity of biological matrices, the analysis of target substances in these samples presents significant challenges to sample processing. To meet these emerging demands on biopharmaceutical analysis, this paper summarizes several new techniques of on-line biological sample processing: solid phase extraction, solid phase micro-extraction, column switching, limited intake filler, molecularly imprinted solid phase extraction, tubular column, and micro-dialysis. We describe new developments, principles, and characteristics of these techniques, and the application of liquid chromatography–mass spectrometry (LC–MS) in biopharmaceutical analysis with these new techniques in on-line biological sample processing.

Pharmacokinetic study of benfotiamine and the bioavailability assessment compared to thiamine hydrochloride

Benfotiamine is a lipid‐soluble thiamine precursor which can transform to thiamine in vivo and subsequently be metabolized to thiamine monophosphate (TMP) and thiamine diphosphate (TDP). This study investigated the pharmacokinetic profiles of thiamine and its phosphorylated metabolites after single‐ and multiple‐dose administration of benfotiamine in healthy Chinese volunteers, and assessed the bioavailability of orally benfotiamine administration compared to thiamine hydrochloride. In addition, concentration of hippuric acid in urine which is produced in the transformation process of benfotiamine was determined. The results showed that thiamine and its phosphorylated metabolites exhibited different pharmacokinetic characteristics in plasma, blood and erythrocyte, and one‐compartment model provided the best fit for pharmacokinetic profiles of thiamine. The transformation process of benfotiamine to thiamine produced large amount of hippuric acid. No accumulation of hippuric acid was observed after multiple‐dose of benfotiamine. Compared to thiamine hydrochloride, the bioavailability of thiamine in plasma and TDP in erythrocyte after oral administration of benfotiamine were 1147.3 ± 490.3% and 195.8 ± 33.8%, respectively. The absorption rate and extent of benfotiamine systemic availability of thiamine were significantly increased indicating higher bioavailability of thiamine from oral dose of benfotiamine compared to oral dose of thiamine hydrochloride.

Intracellular pharmacokinetic study of zidovudine and its phosphorylated metabolites

Zidovudine (AZT), the first drug approved by the US Food and Drug Administration for the treatment of human immunodeficiency virus (HIV) infection, is metabolized in the host cells to 5′-AZT triphosphate (AZT-TP) which inhibits HIV reverse transcriptase. As the pharmacokinetics of AZT and its phosphorylated metabolites in human peripheral blood mononuclear cells (hPBMCs) is limited, the aim of this study was to determine the pharmacokinetic parameters of AZT and its phosphorylated metabolites in hPBMCs from 12 healthy Chinese male subjects after a single oral dose of 600 mg of AZT. Blood samples were collected prior to drug administration, then at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8 and 10 h after drug administration. Mononuclear cells collected by Ficoll-Hypaque density gradient centrifugation were used for determination of AZT and metabolites [AZT monophosphate (AZT-MP), AZT diphosphate (AZT-DP) and AZT-TP] and the plasma was used to evaluate the pharmacokinetics of AZT. Plasma concentration of AZT peaked within 0.583 h and intracellular concentrations of AZT, AZT-MP, AZT-DP and AZT-TP peaked within 1.083, 1.500, 1.417 and 1.583 h, respectively. AZT in plasma was eliminated rapidly with t1/2 of 2.022 h, and AZT-MP, AZT-DP and AZT-TP were eliminated with t1/2 of 13.428, 8.285 and 4.240 h, respectively. The plasma concentration of the phosphorylated metabolites was not quantifiable.

Development and validation of a simple HPLC assay for the quantitation of SM-1, a novel derivative of the PAC-1 anticancer agent, and an initial pharmacokinetics study in rats

A simple and specific HPLC-UV method was developed and validated for the determination of SM-1 in rat plasma. The bioanalytical procedure involved extraction of SM-1 and gefitinib (internal standard, IS) from a 100 μL plasma aliquot with simple protein precipitation with methanol. Chromatographic separation was achieved using an Agilent TC-C18 column (4.6 mm × 150 mm, 5 μm) with an isocratic mobile phase consisting of 10 mM potassium hydrogen phosphate solution (pH 7.0, adjusted by using 10% phosphoric acid)–acetonitrile (37 : 63, v/v) at a flow-rate of 1.0 mL min−1. The UV detection wavelength was 282 nm. SM-1 and IS eluted at 3.6 and 7.0 min, respectively, with a total run time of 8 min. Method validation was performed according to US Food and Drug Administration bioanalytical guidelines and the results met the acceptance criteria. The calibration curve of SM-1 in rat plasma was linear over the concentration range of 0.1–20 μg mL−1. Intra- and inter-run precisions of SM-1 were less than 6.1% and the biases were within ±10.0%. The method was successfully applied to the pharmacokinetics study after intravenous and oral administration of SM-1 in rats.

Implementation of a reference-scaled average bioequivalence approach for highly variable generic drug products of agomelatine in Chinese subjects.

The aim of this study was to apply the reference-scaled average bioequivalence (RSABE) approach to evaluate the bioequivalence of 2 formulations of agomelatine, and to investigate the pharmacokinetic properties of agomelatine in Chinese healthy male subjects. This was performed in a single-dose, randomized-sequence, open-label, four-way crossover study with a one-day washout period between doses. Healthy Chinese males were randomly assigned to receive 25 mg of either the test or reference formulation. The formulations were considered bioequivalent if 90% confidence intervals (CIs) for the log-transformed ratios and ratio of geometric means (GMR) of AUC and Cmax of agomelatine were within the predetermined bioequivalence range based on RSABE method. Results showed that both of the 90% CIs for the log-transformed ratios of AUC and Cmax of 7-desmethyl-agomelatine and 3-hydroxy-agomelatine were within the predetermined bioequivalence range. The 90% CIs for natural log-transformed ratios of Cmax, AUC0–t and AUC0–∞ of agomelatine (104.42–139.86, 101.33–123.83 and 97.90–117.94) were within the RSABE acceptance limits, and 3-hydroxy-agomelatine (105.55–123.03, 101.95–109.10 and 101.72–108.70) and 7-desmethyl-agomelatine (104.50–125.23, 102.36–111.50 and 101.62–110.64) were within the FDA bioequivalence definition intervals (0.80–1.25 for AUC and 0.75–1.33 for Cmax). The RSABE approach was successful in evaluating the bioequivalence of these two formulations.

A sensitive LC-ESI-MS/MS method for the determination of clotrimazole in human plasma

A rapid, sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of clotrimazole in human plasma was developed and validated. The plasma samples were extracted with a mixed solvent of methyl tert-butyl ether–dichloromethane (4 : 1, v/v), and estazolam was selected as the internal standard. Then, the separation was carried out on a Phenomenex Luna CN column (2.0 × 150 mm, 5 μm), using a mobile phase of methanol-0.1% aqueous formic acid solution (85 : 15, v/v). A triple quadrupole mass spectrometer with the positive ionization mode was used for the determination of target analytes. The monitored ion transitions were m/z 276.9 → 165.1 for clotrimazole and m/z 294.9 → 266.9 for estazolam, respectively. The calibration curve of clotrimazole was established over the range of 0.01563–1.000 ng mL−1 (r2 = 0.9935). The intra- and inter-day precisions were less than 10% and all the biases were not more than 9%. The mean extraction recovery of clotrimazole was greater than 68.4% and no significant matrix effect was detected. The LLOQ of 0.01563 ng mL−1 is sensitive enough to perform pharmacokinetic studies after clotrimazole administration.

Determination of Intracellular Concentrations of Nucleoside Analogues and their Phosphorylated Metabolites

Nucleoside analogues are broadly used in antiviral and anti-tumor therapy. The clinical response depends on the intracellular formation of the pharmacologically active mono-, di-, and tri-phosphates moiety. So it is not advisable to simply monitore plasma concentration without concerning the variation of concentration of active constituent in some particular effector cell apparently while this kind of drugs are applied to the clinical therapy. Therefore, determination of intracellular concentration of nucleoside analogues and their phosphorylated metabolites is of great significance. In this review, several methods including RP-HPLC, LC-MS/MS, radioimmunoassay and capillary electrophoresis (CE) for analysis of nucleoside analogues are discussed. Because of the complex biological matrices as well as the extremely low concentration of target analytes, cell lysis techniques and sample pretreatment methods such as protein precipitation (PP) and solid-phase extraction (SPE) are also discussed.

LC-ESI-MS/MS determination of simotinib, a novel epidermal growth factor receptor tyrosine kinase inhibitor: Application to a pharmacokinetic study

Simotinib is a novel epidermal growth factor receptor tyrosine kinase inhibitor. This study presented a sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry method using erlotinib as internal standard for the determination of simotinib in human plasma. The method involved a simple liquid-liquid extraction using diethyl ether. The analytes were separated with isocratic gradient elution on an Agilent TC-C18 column (4.6 × 150 mm, 5 μm). Mass spectrometric detector equipped with electrospray ionization source was carried out in the mode of multiple reaction monitoring (MRM). The monitored transitions were m/z 501.2→182.1 for simotinib and m/z 394.4→278.1 for erlotinib. The calibration curve of simotinib was established over the range of 2.058-3000 μg L(-1) (r(2)=0.9924). The intra- and inter-day precisions were all less than 10%, and all the biases were not more than 7%. This validated method was then successfully applied to a pharmacokinetic study involving twelve healthy Chinese volunteers. The mean Cmax and Tmax for simotinib were 254.79±98.30 μg L(-1) and 1.71±0.48 h, respectively. Plasma concentrations declined with a t1/2 of 5.37±2.32 h. AUC0-t and AUC0→∞ values obtained were 1262.59±501.41 μg L(-1) h and 1329.95±517.42 μg L(-1) h, respectively.

Determination of Strong Acidic Drugs in Biological Matrices: A Review of Separation Methods

Strong acidic drugs are a class of chemical compounds that normally have high hydrophilicity and large negative charges, such as organophosphatic compounds and organosulphonic compounds. This review focuses on sample preparation and separation methods for this group of compounds in biological matrices in recent years. A wide range of separation techniques, especially chromatographic method, are presented and critically discussed, which include liquid chromatography (e.g., ion-pair and ion-exchange chromatography), capillary electrophoresis (CE), and other types. Due to the extremely low concentration level of target analytes as well as the complexity of biological matrices, sample pretreatment methods, such as dilute and shoot methods, protein precipitation (PP), liquid-liquid extraction (LLE), solid-phase extraction (SPE), degradation, and derivatization strategy, also play important roles for the development of successful analytical methods and thus are also discussed.