Drug monitoring and toxicology: a simple procedure for the monitoring of felbamate by HPLC-UV detection

This article describes a simple isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the determination of felbamate in the serum of patients with epilepsy. Sample preparation requires only protein precipitation with a single-step methanol extraction. After centrifugation, the resulting supernatant was injected directly onto the HPLC system. Separation was achieved by reversed-phase HPLC, using a 5-microm Microsorb-MV C(18) column (250 x 4.6 mm) connected to a Silica C(18) guard column (20 x 4.6 mm) and a mobile phase consisting of a mixture of phosphate buffer (pH = 6.9, 0.05 M), methanol, and acetonitrile (64:18:18, v/v/v). The flow rate was at 1.0 mL/min and column temperature was set at 35 degrees C. Quantitation was performed by measurement of the UV absorbance at a wavelength of 210 nm. Calibration curves were linear over a range of 2-200 mg/L, which covered the proposed range of 50-150 mg/L for reference. Both within-run and between-run precision were lower than 5%. Recoveries ranged between 97% and 105% for spiked and pooled samples. No interferences with other common antiepileptic drugs (except zonisamide) were observed. The method requires only 100 microL of serum or less. It is simple and fast (sample preparation and analysis time) and suitable for routine clinical use.     

Drug monitoring and toxicology: a procedure for the monitoring of levetiracetam and zonisamide by HPLC-UV

This article describes a rapid isocratic high-performance liquid chromatographic (HPLC) method for the simultaneous measurement of the anticonvulsants levetiracetam and zonisamide. Monitoring these drugs is important for detecting potentially toxic concentrations, particularly in patients with renal impairment, but no commercial assays are currently available. Following a liquid-liquid extraction, levetiracetam (5-150 microg/mL) and zonisamide (5-80 microg/mL) are quantitated by HPLC-UV. The assay's limit of quantitation, linearity, imprecision, and accuracy adequately cover the therapeutic range of these drugs. The assay should be attractive to clinical laboratories because the run time for quantification of both drugs is approximately 5 min per sample, and no interferences are currently known.     

A rapid punishment procedure for detection of anxiolytic compounds in mice

Rationale Effects of compounds on punished responding have been predictive of anxiolytic efficacy in humans. The use of mice in these tests has been limited, but the utility of this species in drug discovery and for neurobiological inquiry would benefit from a rapid, reliable method.Objectives The present experiments were designed to validate a new procedure in mice.Methods Male, NIH Swiss mice were food deprived and placed in an experimental chamber with two nose-poke holes. Every nose poke (FR1) produced a 20 mg food pellet. On the following day, a drug vehicle was administered and the mice were again exposed to the FR1 schedule. On day 3, a compound was given and the mice were run under a mixed FR1 (food), FR1 (food+shock) schedule in alternating, unsignalled periods of 4 and 10 min for three cycles. In the 10-min periods, nose-pokes produced both food plus brief electrification of the grid floor (0.5 mA for 100 ms). Effects of compounds on food intake were also evaluated in separate groups of mice.Results The introduction of shock substantially decreased responding during the 10-min punishment periods without significantly affecting responding during the non-punishment periods. The clinically effective anxiolytic agents chlordiazepoxide, pentobarbital, and bretazenil, but not buspirone, produced dose-dependent increases in suppressed responding, whereas d-amphetamine, chlorpromazine, and morphine were not effective. Chlordiazepoxide and bretazenil increased food consumption.Conclusions The present method enables rapid and reliable evaluation of potential anxiolytic agents in mice with minimal training. Increases in food intake are not necessary for anxiolytic-like effects under these conditions.     

A rapid HPLC-UV method for the quantification of erythromycin in dermatological preparations

According to the USP erythromycin determination in finished oral products as well as in some topical formulations is mainly carried out via microbiological assays. However, these assays are known for their long incubation periods, lack of precision and low sensitivity. In literature one HPLC method for the quantification of erythromycin in creams is described, which depends on electrochemical detection, but HPLC electrochemical detection has not emerged as popular choice in routine analysis. Furthermore two other HPLC-UV methods are described for the isolation of erythromycin from gels and creams involving tedious and time consuming extraction steps, the reason why they are not suited to be applied in routine analysis. This paper describes a new HPLC-UV method for the determination of erythromycin in creams, which implies a much easier extraction procedure than that cited in literature to date, based solely on the solubilization of erythromycin followed by freezing the cream matrix. Validation experiments confirmed the precision and accuracy of the method. Good linearity of the assay was found over the investigated concentration range of 70-130% (corresponding to 0.77-1.43 g of erythromycin A in 100 g cream base). The coefficient of correlation resulting from unweighted linear regression was 0.9998, allowing a one-point calibration in routine analysis. By the implementation of an internal standard in the quantification of erythromycin an improved precision could be achieved in routine analysis. This new analytical method yields cleaner extracts and allows a higher throughput, saving costs, solvents and time and can be thus recommended to all laboratories.     

Tissue extraction of amiodarone and N-desethylamiodarone in man after a single oral dose.

The hepatic extraction of amiodarone and N-desethylamiodarone has been investigated in seven patients following catheterization of the portal and hepatic veins under general anaesthesia. Amiodarone (600 mg) was administered orally 4 h before regional blood sampling. Concentrations of amiodarone and N-desethylamiodarone, determined by h.p.l.c., were about twice as high in the portal vein compared with those in the hepatic vein, the calculated hepatic extraction ratios of both compounds being 0.39 +/- 0.07 and 0.34 +/- 0.03, respectively. The presence of N-desethylamiodarone in the portal vein in higher concentrations than in the hepatic vein strongly suggests that N-dealkylation of amiodarone occurs in the gut wall or lumen, a finding which might account for the low and highly variable intersubject amiodarone bioavailability.     

A rapid electron-capture gas chromatographic procedure for the analysis of p-hydroxymephenytoin

An electron-capture gas chromatographic procedure was developed for detection and quantification of p-hydroxymephenytoin (OHMEP), a metabolite of S-mephenytoin, in human liver microsomal preparations. OHMEP was derivatized with pentafluorobenzoyl chloride (PFBC) under basic aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column and an electron-capture detector. Dextrorophan was carried through the procedure as internal standard. The structure of the PFB derivative was confirmed using combined gas chromatography-mass spectrometry (GC-MS). The procedure is rapid and reproducible and produces a stable derivative that has excellent chromatographic properties. The limit of detection was less than 5 ng/ml, and the method was applied to extracts of human liver microsomes, which had been incubated with S-mephenytoin [a probe substrate for cytochrome P450 (CYP) 2C19].     

A rapid microbiotest for the detection of cyanobacterial toxins

Cyanobacteria occur widely in lakes, reservoirs, ponds, and slow flowing rivers. Many species are known to produce toxins (cyanotoxins), a number of which are of concern for health. Cyanotoxins vary in chemical structure and may be found intracellular or released into water. There is not only a wide variation in the toxicity of known cyanotoxins but a substantial number of toxins have to date not been identified chemically. Chemical analysis of cyanotoxins is nowadays not used for routine monitoring because it is time consuming, it requires specialized equipment and expertise, and is hence expensive. There is hence an urgent need for rapid tests in surface waters to detect cyanobacterial toxins because of the need for safe drinking water and safe natural bathing waters, which may be burdened by cyanobacterial blooms or scums. Previous investigations have already shown that larvae of the anostracan crustacean Thamnocephalus platyurus are quite sensitive to neurotoxic and hepatotoxic cyanotoxins. The present paper reports on the sensitivity comparison of the (1 h) Rapidtoxkit (based on a sublethal endpoint) and the (24 h) Thamnotoxkit microbiotest (based on mortality). Both assays make use of larvae of T. platyurus. The Rapidtoxkit is a new microbiotest that determines the decrease of ingestion of colored particles by the crustacean larvae, which are stressed by a short exposure to toxicants. Fifteen cyanobacterial samples composed of laboratory strains and natural bloom samples were tested by both microbiotests. All samples were also analyzed concurrently by HPLC for microcystins and cylindrospermopsin. The correlation coefficient between the two microbiotests (r = 0.82) showed the very good correspondence between the sublethal and the lethal effects. No known toxins could be detected in some samples, although the latter were found highly toxic to the test organisms in both bioassays. These results point to the presence of unknown toxin(s) produced by some cyanobacteria such as e.g., the Cylindrospermopsis raciborskii strain isolated from Lake Balaton in Hungary. This comparative study clearly showed that the 1 h Rapidtoxkit is an attractive rapid alternative to the Thamnotoxkit microbiotest.     

A rapid and simple procedure for the determination of synephrine in dietary supplements by gas chromatography-mass spectrometry

A simple and rapid procedure based on gas chromatography-mass spectrometry (GC-MS) is described for determination of synephrine, active principle of Citrus aurantium plant, in solid and liquid dietary supplements. After the addition of 3,4-methylenedioxypropylamphetamine as internal standard (I.S.), a liquid-liquid extraction procedure in alkaline conditions with chloroform/isopropanol (9:1, v/v) was applied to the samples prior to analysis. Chromatography was performed on a fused capillary column and synephrine and I.S., derivatized with pentafluoropropionic anhydride, were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 0.1-50 microg/mg or microg/mL synephrine. Mean recovery ranged between 89.3% and 90.5% in both solid and liquid dietary supplements. The quantification limit was 0.1 microg/mg or microg/ml. The method was applied to analysis of various dietary supplements promoted for aiding weight control containing, among other constituents such as ephedrine alkaloids and methylxanthines, Citrus aurantium. Amount of synephrine present in such products ranged from 3.1 microg/mg solid product to 480.2 microg/mL liquid product.     

A rapid procedure to prepare cefotaxime

A rapid procedure is reported for the synthesis of cefotaxime, by acylation of the 7-amino cephalosporanic acid with the 2-mercaptobenzothiazolyl thioester of (Z)-2-[2-aminothiazol-4-yl]-2-methoxyimino acetic acid (MAEM) that is a commercial reagent. The reaction was carried out at room temperature for 1 h, obtaining 95% yield. 2-Mercaptobenzothiazole was recovered as a side-product with a high purity and yield. The proposed method differentiates from those reported previously for a shorter time and very mild reaction condition, as well as for a ready for use reagent.     

HPLC法同时测定人血浆中的伪麻黄碱和氯苯那敏

目的建立同时测定血浆中盐酸伪麻黄碱、马来酸氯苯那敏的方法。方法采用反相HPLC法,以右美沙芬为内标,血浆中的被测药物经甲基叔丁基醚提取、1.5%盐酸溶液反萃后同时测定。色谱柱:C₁₈(250 mm×4.6 mm ID,5 μm);流动相:乙腈-水-三乙胺(46∶54∶0.2,内含10 mmol·L^-1十二烷基硫酸钠,60 mmol·L^-1磷酸二氢钠,以磷酸调pH为2.6);检测波长:200 nm。结果伪麻黄碱、氯苯那敏的线性范围分别为1.5～0.01 mg·L^-1和75.0～0.5 μg·L^-1, 检测限分别为10.0和0.5 μg·L^-1;日内、日间RSD小于12.4%,方法平均回收率为97.3%～109.4%。结论该法简便、快速,重现性好,灵敏度高,可用于盐酸伪麻黄碱、马来酸氯苯那敏复方制剂的临床药代动力学研究。     

A rapid and simple procedure for the determination of ephedrine alkaloids in dietary supplements by gas chromatography-mass spectrometry

A simple method for the determination of ephedrine alkaloids: ephedrine (EF), pseudoephedrine (PE), norpseudoephedrine (NPE), norephedrine (NE) and methylpseudoephedrine (MPE) in dietary supplements by gas chromatography–mass spectrometry is described. After the addition of 3,4-methylenedioxypropylamphetamine as internal standard, a liquid–liquid extraction procedure in alkaline conditions with chloroform/isopropanol (9:1, v/v) was applied to the samples prior to analysis. Chromatography was performed on a fused capillary column and analytes, derivatized with pentafluoropropionic anhydride, were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 0.3–10 μg/mg for EP, 0.06–2.5 μg/mg for PE and NPE and 0.04–1 μg/mg NE and MPE. Mean recovery ranged between 65.7 and 81.3% for the different analytes in dietary supplements. The quantification limits were 0.3 μg/mg for EP, 0.06 μg/mg for PE, 0.04 μg/mg for NPE, NE and MPE. The method was applied to analysis of various dietary supplements containing Ma-huang (Ephedra Sinica) and Sida Cordifolia plant extracts promoted for aiding weight control and boosting sports performance and energy.     

A rapid and simple procedure for the determination of cannabinoids in hemp food products by gas chromatography-mass spectrometry

A rapid and simple procedure using liquid–liquid extraction and subsequent gas chromatographic mass-spectrometric detection has been developed for determination of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in different hemp foods. After addition of Δ⁸-tetrahydrocannabinol as internal standard, both solid and liquid specimens were extracted with two volumes of 2 ml of hexane/isopropanol (9:1): Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 1–50 ng/ml liquid samples or 1–50 ng/g solid samples for THC and CBN, and 2–50 ng/ml or ng/g for CBD. Mean recoveries ranged between 78.8 and 90.2% for the different analytes in solid and liquid samples. The quantification limits were 1 ng/ml or ng/g for THC and CBN and 2 ng/ml or ng/g CBD. The method was applied to analysis of various hemp foods. THC content in different products varied 50-fold, whereas CBN and CBD were absent in some samples and achieved hundreds of ng/ml or ng/g in others. The concentration ratio (THC + CBN)/CBD was used to differentiate between the phenotypes of cannabis plants in different specimens. Products possibly originating from drug-type cannabis plants were found in the majority of analyzed specimens.     

A rapid one-step immunochromatographic assay for the detection of asiaticoside

Asiaticoside has been identified as the most active compound in Centella asiatica. In order to screen a large number of plant samples for the presence of asiaticoside, a rapid and simple technique is required that utilizes small quantities for test samples. In this study, an immunochromatographic strip test has been developed for the detection of asiaticoside in plant samples that uses a monoclonal antibody against asiaticoside. The limit of detection for the strip test was 12.5 μg/ml. Immunoassay using monoclonal antibodies could be useful for the determination of small quantities of asiaticoside in plant extracts.     

A SPME-GC procedure for monitoring peppermint flavor in tablets

A method was developed using solid-phase microextraction (SPME) and gas chromatography to monitor the peppermint flavor loss in a taste-masked tablet formulation. This was accomplished by headspace sampling of two major components of peppermint: menthone and menthol. It was found that the excipients from the tablet produced an important matrix effect and that standard addition analysis was necessary for improved accuracy of the determination. The method was shown to be specific and precise. Furthermore, the method produced acceptable results with adequate quantitation limits to determine peppermint flavors in taste-masked tablets. The optimized extraction procedure was successfully used to monitor the stability of peppermint flavor in an oral solid formulation. The accelerated stability studies of the tablet showed that the menthone and menthol was lost in an exponential manner and levels off after several days of heat exposure.     

A rapid screening procedure for acidic and neutral drugs in blood by high resolution gas chromatography

A rapid high resolution gas chromatographic method for screening acidic and neutral drugs in blood is described. The procedure involves a single extraction with ethyl acetate. Using flame ionization detection, without derivatization and with on-column methylation, more than 60 drugs of toxicologic importance are detected.     

A non-extracting procedure for the determination of meloxicam in plasma samples by HPLC-diode array detection

The bioequivalence of two formulations of 15 mg tablets of meloxicam (CAS 71125-38-7), Meloxicam (LaborMed Pharma) and a commercially available preparation as reference in 24 healthy male and female Caucasian volunteers was assessed based on a validated non-extractive HPLC-DAD method. The sample preparation procedure was based on protein precipitation with a mixture of methanol/acetonitrile, trifluoacetic acid and sodium sulfate solution. Piroxicam (CAS 36322-90-4) was used as internal standard. The stepwise gradient elution profile of the chromatographic method allows injection of a high volume of the sample (500 microL) with the focusing of both analytes in a Chromolith Performance RP-18e column. The mobile phase constituents are methanol and aqueous 20 mmol/L Na2HPO4 buffer solution brought to pH = 6 with H3PO4. A flow-rate of 2 mL/min achieves a complete chromatographic run (including column equilibration) within 12 min. UV detection at 356 nm allowed a quantitation limit around 30 ng/mL. Bioequivalence study was based on an open-label, randomized, two-period, two-sequence, single dose, crossover design with a 2-week wash-out period between consecutive oral administrations. The main pharmacokinetic parameters (C(max), t(max), t 1/2, AUD and AUC(0-infinity)) were considered as evaluation criteria for the bioequivalence of the test drug against the reference.