Estimation of plasma lorazepam by gas‐liquid chromatography and a benzene extraction

Abstract

Lorazepam, a new long-acting benzodiazepine, has been in clinical use for about one year. As part of its evaluation as a premedicant it was necessary to correlate plasma levels with clinical assessment after various modes of administration. A method of extracting lorazepam from plasma was obtained from the manufacturers but proved to be unsuitable when large numbers of samples had to be analysed. It involved a number of stages. A similar method was reported by Knowles, Comer and Ruelius. 'In 1975 Gamble and his colleagues2 described a benzene extraction of diazepam from plasma and from this a method of extraction and estimation of lorazepam was evolved. The method involves a single stage benzene extraction with flunitrazepam as the internal standard which allows 40 samples to be extracted in approximately 2+ hours and chromatographed in 5 hours. The method is described in this paper with critiques on its reliability.The blood samples analysed were obtained during the clinical studies reported by Dundee and c011eagues~~~ and Lilburn, Dundee and Moore5 including the long term use of lorazepam.Extraction. 0.5 ml plasma was extracted with 5 ml of 'Ultrar' grade benzene to which 0.5 ml potassium chloride (Analar) and 50 pl of a 150 ng/ml solution of flunitrazepam in benzene had been added. The mixture was shaken for 15 min on a rotary mixer at 45 revlmin and then centrifuged at 4 000 revlmin for 5 min. Four ml of the benzene layer were removed and evaporated to dryness, reconstituted with 50 pl of benzene and 10 , d injected on to the column.A Perkin Elmer F 33 gas chromatograph fitted with a 63Ni electron capture detector (ECD) was used. The column was of borosilicate glass 120 cm in length with an internal diameter of 0.5 cm and packed with 3% OV17 on gas chrom Q 80-100 mesh.Injection temperature 300°C ECD setting 4 Detector temperature 300°C Attenuation 32 Oven temperature 240°C Range 1The carrier gas was oxygen free nitrogen (OFN) at a pressure of 350 kN/mZ. Under these conditions the retention time of lorazepam was 1.9 min and 3.6 min for the internal standardflunitrazepam (Fig. 1). Estimation. A calibration curve is prepared from lorazepam concentrations of 0,25, 50,75, 100 and 125 ng/ml in plasma. The ratio of the lorazepam peak (H) to the internal standard peak (HS) is calculated and the mean of each set of duplicate samples plotted against the lorazepam concentrations. Figure 2 is a typical calibration curve.All observations were carried out in duplicate. From a random batch of 197 plasma lorazepam estimations the duplicate individual levels were Conditions of analysis are:

Determination of lorazepam in plasma from children by high-performance liquid chromatography with UV detection

“…Analytical methods for LZP in biological fluids are problematic, due to the high sensitivity required to detect the low unconjugated blood (plasma/serum) LZP concentrations achieved after the recommended doses, and the thermal instability of the 3-hydroxybenzodiazepine ring. Previously published GC methods for the quantification of LZP and/or conjugated glucuronide metabolites in biological matter (whole blood, plasma/serum urine, or brain) include GC-ECD [19][20][21][22]45]; GC-NPD [19,23]; GC-MS [25,26] and GC-MS/NICI [27,28] or GC-MS/SIM [24,28,30]. Several HPLC methods with UV or diode array detection [31][32][33][34][35][36][37][38][39][40][41]43] and mass spectrometric detection [43] have also been reported.…”

Pharmacokinetics of Diazepam and Midazolam When Used for Sedation Following Cardiopulmonary Bypass

“…as required. Venous blood was drawn from both groups at 0, 7.5, 15, 30, 60 and 90 min and at 2, 4, 6, 8, 10 and 12 h after the initial dose, and the plasma concentrations of each benzodiazepine measured (gas-liquid chromatography) (Gamble et al, 1975;Howard et al, 1978). (The coefficient of variation of our analyses was 3.4% for diazepam, 9?8 % for low concentrations of midazolam and 1.8% for high concentrations.)…”

The effect of intravenous cimetidine on the absorption of orally administered diazepam and lorazepam.

“…Plasma lorazepam and diazepam estimations were by gas-liquid chromatography (Howard et al, 1977;Gamble et al, 1975). Cimetidine does not interfere with either of these determinations.…”