Reversed-phase ion-pair HPLC method for the direct analysis of 1-OH midazolam glucuronide in human serum.

In recent years, it has become clear that the presence of high concentrations of 1-OH midazolam glucuronide is probably the cause of unexplained prolonged midazolam comas in patients with poor renal function. Until recently, only indirect methods for the analysis of this glucuronide were known, which had several disadvantages, such as a long analysis period (>6 hours). This article describes the validation of a method for the direct analysis of this compound in human serum, using reversed-phase ion-pair high-performance liquid chromatography (HPLC) in combination with solid phase extraction. The intraday and interday coefficients of variation have values below 6% for different possible serum concentrations. The limit of quantification (0.1 mg/L) is much lower than concentrations found in patients with a coma caused by the accumulation of 1-OH midazolam glucuronide. Recovery of 1-OH midazolam glucuronide is almost 100% at three different serum concentrations. Linearity is confirmed for normal serum levels (<1 mg/L) and for serum levels that might occur in patients with impaired renal function (<20 mg/L). Detection is performed at 254 nm with a diode array detector, which can also be used to check the peak purity in case of unexpected impurities.

Rapid method for the analysis of itraconazole and hydroxyitraconazole in serum by high-performance liquid chromatography.

An assay for the determination of itraconazole and its active metabolite hydroxyitraconazole in serum has been developed, using ketoconazole as internal standard. The procedure involved a one-step liquid-liquid extraction of the drug, its metabolite and the internal standard, followed by their separation by reversed-phase HPLC. In this paper the validation of this method is described.

Determination of 4-aminopyridine in serum by solid-phase extraction and high-performance liquid chromatography.

An assay for the determination of 4-aminopyridine in serum has been developed using 3,4-diaminopyridine as internal standard and reversed-phase high-performance liquid chromatography with detection at 244 nm. A mobile phase of acetonitrile-methanol-ethanol-1% ammonium carbonate (75:10:10:5) provided excellent separation of both compounds. Samples were extracted on solid-phase columns. The linearity, precision, recovery and the limit of detection were all sufficient for the routine use of this assay in clinical studies of patients treated with 4-aminopyridine.

Sorption of various drugs in polyvinyl chloride, glass, and polyethylene-lined infusion containers.

The sorption of chloroquine sulfate, diazepam, isosorbide dinitrate, lorazepam, midazolam, nitroglycerin, promethazine hydrochloride, thiopental sodium, and warfarin sodium to three types of containers was studied. Appropriate amounts of the drugs were added to 500 mL of 0.9% sodium chloride injection in polyvinyl chloride (PVC) bags, glass bottles, and Clear-Flex bags composed of a laminate of polyethylene, nylon, and polypropylene. The containers were stored in the dark at room temperature for 24 hours. Samples were taken at various intervals and assayed for drug concentration by high-performance liquid chromatography. There were no appreciable changes in pH after 24 hours, and all the admixtures remained clear and colorless. The potency of chloroquine sulfate, lorazepam, midazolam, promethazine hydrochloride, and thiopental sodium remained unchanged in glass, PVC, and Clear-Flex containers. Diazepam, isosorbide dinitrate, nitroglycerin, and warfarin sodium did not show any sorption to glass bottles and Clear-Flex bags. In PVC bags, however, up to 55% of diazepam, 23% of isosorbide dinitrate, 51% of nitroglycerin, and 24% of warfarin sodium was lost during the 24-hour study period. Diazepam, isosorbide dinitrate, nitroglycerin, and warfarin sodium in 0.9% sodium chloride injection showed a loss of potency when stored in PVC containers for 24 hours at room temperature, but none of the drugs studied lost potency when stored in glass bottles and Clear-Flex bags.

Analysis of water-soluble vitamins in total parenteral nutrition solution by high pressure liquid chromatography.

A stability-indicating high pressure liquid chromatographic method was developed to determine the stability of water-soluble vitamins in total parenteral nutrition mixtures. Folic acid and thiamine were determined by direct injection and precolumn enrichment, followed by UV detection. Nicotinamide and pyridoxine were determined simultaneously without sample pretreatment by UV detection of nicotinamide and fluorescence detection of pyridoxine. Riboflavin 5'-phosphate was also determined without elaborate sample handling and by fluorescence detection. Ascorbic acid was determined as the sum of ascorbic acid and dehydroascorbic acid. After derivatization to a quinoxaline the latter substance was determined by direct injection and fluorescence detection.

Simultaneous determination of midazolam and its three hydroxy metabolites in human plasma by electron-capture gas chromatography without derivatization.

Electron-capture gas chromatography was carried out to determine midazolam and its three hydroxy metabolites (1-hydroxymethylmidazolam, 4-hydroxymidazolam and 1-hydroxymethyl-4-hydroxymidazolam) in human plasma. The assay involves extraction from plasma, buffered to pH 9.3, into cyclohexane-dichloromethane (6:4) and analysis by gas chromatography. The use of an HP-17 cross-linked, capillary column makes derivatization unnecessary. The sensitivity of the method was 2-3 ng/ml for midazolam, 1-hydroxymethylmidazolam and 4-hydroxymidazolam, and 20 ng/ml for 1-hydroxymethyl-4-hydroxymidazolam. The extraction recovery of midazolam, 1-hydroxymethylmidazolam, 4-hydroxymidazolam and 1-hydroxymethyl-4-hydroxymidazolam was 99.3 +/- 2.4, 67.0 +/- 4.6, 92.7 +/- 4.7 and 28.7 +/- 6.3%, respectively. This gas chromatographic assay was used to assess the concentration-time profiles of midazolam and its metabolites in human plasma after rectal and intravenous administration of midazolam.

Determination of some anticonvulsants, antiarrhythmics, benzodiazepines, xanthines, paracetamol and chloramphenicol by reversed phase HPLC.

In this article assays are described for caffeine, theophylline, procainamide, N-acetylprocainamide, quinidine, dihydroquinidine, paracetamol, phenobarbital, phenytoin, carbamazepine, chloramphenicol, oxazepam, temazepam, diazepam, desmethyldiazepam, chlordiazepoxide, desmethylchlordiazepoxide and demoxepam using a uniform working procedure, five (slightly) different mobile phases and one HPLC system. Changing from one eluent to another is simple and a stable base-line is achieved within half an hour. Three of the five eluents are interchangeable and recycling of eluent causes no problems. Sample pretreatment is a single step extraction. Interferences can be overcome by changing the selectivity of the eluent by adjusting the tetrahydrofuran or triethylamine content. Furthermore it is shown how triethylamine can improve peak shapes of basic components and shorten their retention times.