Simultaneous Extraction of Selected Benzodiazepines and Benzodiazepine-Glucuronides from Urine by Immunoadsorption

Abstract

A rapid and selective cleanup procedure based on immunoadsorption is described for the simultaneous extraction of diazepam and its free or glucuronidated metabolites nordiazepam, temazepam, and oxazepam from urine. The method can also be used for the extraction of lorazepam and lorazepam-glucuronide. Because the samples do not have to be hydrolyzed before extraction, valuable information is preserved. With the exception of lorazepam-glucuronide, recoveries between 86 and 100% were obtained at spiking levels up to 200 ng of benzodiazepine or glucuronide per milliliter of urine. Using methanol/water (90:10, v/v) as an eluent, the immunoadsorber could be used at least 20 times. High-performance liquid chromatograms of urine samples from patients receiving low therapeutic dosages of diazepam or lorazepam are shown to demonstrate the high purity of the extracts.

Signal enhancement of glucuronide conjugates in LC‐MS/MS by derivatization with the phosphonium propylamine cation tris(trimethoxyphenyl) phosphonium propylamine, for forensic purposes

“…[3,8,[12][13][14][15] Derivatization in urine, however, poses substantial challenges as 55 mmol/day and 64 mmol/day of small organic acids are excreted in women and men, respectively, with Table 1. The approach would be much more applicable to targeted analysis following extensive clean-up employing methods such as immunoadsorption of benzodiazepines and their glucuronides, as reported by Franzelius et al [17] or molecularly imprinted polymers (MIPS). Data was obtained following LC-MS/MS (collision energy 35%) and signal improvements are based on extracted ion chromatograms of the characteristic MS/MS glucuronic acid (excluding any conjugates) accounting for 2 mmol.…”

“…If the volume of urine eliminated in a day is assumed to be 1 L, at 1 ng/mL of benzodiazepine glucuronide, this would correspond to a 3 × 10 7 fold excess of other organic acids. The approach would be much more applicable to targeted analysis following extensive clean-up employing methods such as immunoadsorption of benzodiazepines and their glucuronides, as reported by Franzelius et al [17] or molecularly imprinted polymers (MIPS). [18] Purification, at least partial, is therefore necessary if the analyte is encountered in small concentrations.…”

Antibody-Functionalized Nano Test Tubes Target Breast Cancer Cells

“…The aldehyde groups were then reacted using Schiff-base chemistry to free amino sites on the antibody [48][49][50]. This was accomplished by dispersing the tubes into a PBS solution (pH 7.4) that contained 0.20 mg/ml antibody and was also 4.0 mM in NaBH 3 CN.…”

HPLC with laser-induced native fluorescence detection for morphine and morphine glucuronides from blood after immunoaffinity extraction

“…This technique has already been successfully applied for on-line or separate purification and extraction in several cases [22][23][24], e.g. for the extraction of estradiol [25], clenbuterol [26], amanitin [27], tetrahydrocannabinol [28,29], benzodiazepines and their metabolites [30,31], LSD [32,33] and morphine and its metabolites [34,35]. We developed a new immunoadsorber for the extraction of morphine and its glucuronides by the immobilisation of specific antibodies against morphine and morphine glucuronides [36] applying the solgel method.…”