Analysis of lorazepam in rat brain using liquid/liquid and solid‐phase extraction in combination with high performance liquid chromatography

Abstract

A method for the determination of lorazepam in rat brain is described using liquid/liquid and solid-phase extraction, followed by high performance liquid chromatography. After addition of chlordiazepoxide as the internal standard, 100 mg brain tissue was homogenized and incubated with alkaline protease. Lorazepam and chlordiazepoxide were extracted three times with toluene. After treatment through a C18-Bond Elut column, lorazepam and chlordiazepoxide were analyzed isocratically on a reversed-phase column with a mobile phase consisting of methanol +0.025 M sodium phosphate buffer (66:34, v/v). The eluted drugs were monitored by their absorption at 240 nm. The sensitivity limit of this method was 10 ng of lorazepam per 100 mg of brain tissue sample. The standard curve was linear over the range of 20 to 200 ng lorazepam. The coefficient of variation for day-to-day precision established by 21 replicate analyses was 4.5 to 13.6%.

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

“…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. Although GC methods are more sensitive (with lower limits of quantification ≤2 ng/ml [28,43]) than HPLC methods for measuring LZP, they involve lengthy sample clean-up procedures, and require derivatization steps [25,26,30] to increase the volatility of LZP, which is thermally unstable under GC conditions [22,46].…”

“…These methods include gas chromatography (GC) with electroncapture detection (GC-ECD) [19][20][21][22], nitrogen-phosphorus detection (GC-NPD) [19,23], mass spectrometry (GC-MS) [24][25][26], and GC with negative ion chemical ionization MS (GC-MS/NICI) [27,28] or selected ion monitoring MS (GC-MS/SIM) [28][29][30]. High-performance liquid chromatography (HPLC) methods for quantification of LZP in biological fluids have also been reported, with the choice of detectors, including UV (HPLC-UV) or diode array detector (HPLC-DAD) [31][32][33][34][35][36][37][38][39][40][41][42], and mass spectrometric detection [43]. However, most of these methods have various limitations, including time-consuming sample clean-up and/or derivatization steps [25,26,30]; use of large sample volumes (≥1 ml) [19,21,24,26,29,31,36,41]; inadequate sensitivity [31,32]; and use of expensive solid phase extraction cartridges [25,[34][35]…”

A fast and sensitive liquid chromatographic–tandem mass spectrometric method for assay of lorazepam and application to pharmacokinetic analysis

“…When HPLC is used for separation of lorazepam with the coexisting species such as its metabolism products, no derivation of the drug is required. Nevertheless, (In these reports [2][3][4][5][6][7][8][9][10][11][12][13][14][15],) either liquid-liquid extraction [2][3][4][6][7][8][9][10][11][12]15] or solid phase extraction [5,13,14] was usually employed to perform matrix removal and analyte pre-concentration prior HPLC separation. The extraction-involved sample pre-treatment procedures could enrich the analyte by several folds even 1-2 orders of magnitude, allowing the HPLC or HPLC/MS to be used for determination of lorazepam in low ppb levels.…”

“…Several methods such as high performance liquid chromatography (HPLC) [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], gas chromatography (GC) [17][18][19][20][21][22], micellar electrokinetic capillary chromatography (MECC) [23], immunoassay [24][25][26], adsorptive-stripping voltammetry [27], gas chromatography/mass spectrometry (GC/MS) [28][29][30][31][32], tandem mass spectrometry (GC/MS/MS) [33] as well as high performance liquid chromatography/mass spectrometry [34,35] have been reported for the determination of lorazepam in bio-samples. Among them, GC and HPLC coupled with various detectors are most widely used.…”