Analysis of lorazepam and its 30-glucuronide in human urine by capillary electrophoresis: evidence for the formation of two distinct diastereoisomeric glucuronides

Lorazepam (LOR) is a 3-hydroxy-1,4-benzodiazepine that is chiral and undergoes enantiomerization at room temperature. In humans, about 75% of the administered dose of LOR is excreted in the urine as its 30-glucuronide. CE-MS with negative ESI was used to confirm the presence of LOR-30-glucuronide in urines that stemmed from a healthy individual who ingested 1 or 2 mg LOR, whereas free LOR could be detected in extracts prepared from enzymatically hydrolyzed urines. As the 30-glucuronidation reaction occurs at the chiral center of the molecule, two diastereoisomers can theoretically be formed, molecules that can no longer interconvert. The stereoselective formation of LOR glucuronides in humans and in vitro was investigated. MEKC analysis of extracts of the nonhydrolyzed urines suggested the presence of the two different LOR glucuronides in the urine. The formation of the same two diastereoisomers was also observed in vitro employing incubations of LOR with human liver microsomes in the presence of uridine 5'-diphospho-glucuronic acid as coenzyme. The absence of other coenzymes excluded the formation of phase I or other phase II metabolites of LOR. Both results revealed a stereoselectivity, one diastereoisomer being formed in a higher amount than the other. After enzymatic hydrolysis using beta-glucuronidase, these peaks could not be detected any more. Instead, LOR was monitored. Analysis of the extracts prepared from enzymatically hydrolyzed urines by MEKC in the presence of 2-hydroxypropyl-beta-CD revealed the enantiomerization process of LOR (observation of two peaks of equal magnitude connected with a plateau zone). The data presented provide for the first time the evidence of the stereoselectivity of the LOR glucuronidation in humans.

Rapid determination of gemcitabine in plasma and serum using reversed-phase HPLC

Gemcitabine (2'2'-difluorodeoxycytidine) is a pyrimidine analog used in the treatment of a variety of solid tumors. After intravenous (i.v.) administration, it is rapidly inactivated to 2'-deoxy-2',2'-difluorouridine (dFdU). A sensitive analytical method for the quantitation of gemcitabine is required for the assessment of alternative dosage and treatment schemes. A rapid and robust RP-HPLC assay for analysis of gemcitabine in human and animal plasma and serum was developed and validated using 2'-deoxyuridine (dU) and 5-fluoro-2'-deoxyuridine (5FdU) as internal standards. It is based on protein precipitation, the use of an Atlantis dC18 column of 100 mm length (inner diameter, 4.6 mm; particle size, 3 microm) and isocratic elution using a 10 mM phosphate buffer, pH 3.0, followed by isocratic elution with the same buffer containing 3% of ACN. For gemcitabine, RSD values for intraday and interday precision were < 4.4 and 5.3%, respectively, the LOQ was 20 ng/mL, and the assay was linear in the range of 0.020-20 microg/mL with an accuracy of > or =89%. The recovery for gemcitabine, dU and 5FdU was 86-98%. The assay was applied to determine gemcitabine levels in plasma samples of patients collected during and shortly after conventional infusion of 25-30 mg/kg body mass (levels: 2.0-18.9 microg/mL) and rats that received lower doses (1.5 mg/kg) via i.v., subcutaneous and oral drug administration (levels: 0.20-2.60 microg/mL). It could also be applied to estimate dFdU levels in human plasma.

Capillary electrophoresis evidence of the stereoselective ketoreduction of mebendazole and aminomebendazole in echinococcosis patients

An assay for the simultaneous determination of the enantiomers of hydroxymebendazole (OH-MBZ) and hydroxyaminomebendazole (OH-AMBZ) together with aminomebendazole (AMBZ) in human plasma is described for the first time. It is based upon liquid-liquid extraction at alkaline pH from 0.5 mL plasma followed by analysis of the reconstituted extract by CE with reversed polarity in the presence of a 50 mM, pH 4.2 acetate buffer containing 15 mg/mL sulfated beta-CD as chiral selector. For all compounds, detection limits are between 0.01 and 0.04 microg/mL, and intraday and interday precisions evaluated from peak area ratios are <6.9 and <8.5%, respectively. Analysis of 39 samples of echinoccocosis patients undergoing pharmacotherapy with mebendazole (MBZ) revealed that the ketoreduction of MBZ and AMBZ is highly stereoselective. One enantiomer of each metabolite (firstly detected peak in both cases) could only be detected. The CE data revealed that OH-MBZ (mean: 0.715 microg/mL) is the major metabolite followed by AMBZ (mean: 0.165 microg/mL) and OH-AMBZ (mean: 0.055 microg/mL) whereas the MBZ plasma levels (mean: 0.096 microg/mL, levels determined by HPLC) were between those of AMBZ and OH-AMBZ.

Determination of carbohydrate-deficient transferrin in human serum by two capillary zone electrophoresis methods and a direct immunoassay: comparison of patient data

Data obtained with two CZE assays for determining carbohydrate-deficient transferrin (CDT) in human serum under routine conditions, the CAPILLARYS CDT and the high-resolution CEofix (HR-CEofix) CDT methods, are in agreement with patient sera that do not exhibit interferences, high trisialo-transferrin (Tf) levels or genetic variants. HR-CEofix CDT levels are somewhat higher compared to those obtained with the CAPILLARYS method and this bias corresponds to the difference of the upper reference values of the two assays. The lower resolution between disialo-Tf and trisialo-Tf observed in the CAPILLARYS system (mean: 1.24) compared to HR-CEofix (mean: 1.74) is believed to be the key for this difference. For critical sera with high trisialo-Tf levels, genetic variants, or certain interferences in the beta-region, the HR-CEofix approach is demonstrated to perform better than CAPILLARYS. However, the determination of CDT with the HR-CEofix method can also be hampered with interferences. Results with disialo-Tf values larger than 3% in the absence of asialo-Tf should be evaluated with immunosubtraction of Tf and possibly also confirmed with another CZE method or by HPLC. Furthermore, data gathered with the N Latex CDT direct immunonephelometric assay suggest that this assay can be used for screening purposes. To reduce the number of false negative results, CDT data above 2.0% should be confirmed using a separation method.

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE-based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4-dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, epsilon-aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs-Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.

An MEKC assay for the therapeutic drug monitoring of cefepime

The development of a robust assay based on MEKC for cefepime in human serum and plasma with internal quality assurance is reported. Sample preparation comprises protein precipitation in the presence of SDS at pH 4.5. This is a gentle approach for which decomposition of cefepime during sample handling is negligible. After hydrodynamic sample injection of the supernatant, analysis occurs in a phosphate/borate buffer at pH 9.1 with 75 mM SDS using normal polarity and analyte detection at 257 nm. The MEKC run time interval and throughput are about 5 min and seven samples per hour, respectively. The calibration range for cefepime is 1-60 μg/mL, with 1 μg/mL being the LOQ. The performance of the assay with multilevel internal calibration was assessed with calibration and control samples. The assay is shown to be simple, inexpensive, reproducible, and robust. It was applied to determine cefepime levels in the sera of critically ill patients and to assess the instability of cefepime in patient and control samples. Our data revealed that serum containing cefepime can be stored at -20°C for a short time, whereas for long-term storage, samples have to be kept at -70°C.

Monitoring of alcohol markers by capillary electrophoresis.

Work dealing with the monitoring of alcohol markers by CE performed during the past two decades led to the development of assays for carbohydrate-deficient transferrin (CDT), ethyl sulfate, ethyl glucuronide, and phosphatidylethanol in body fluids and first attempts for the detection of the urinary 5-hydroxytryptophol/5-hydroxyindoleacetic acid ratio and stable hemoglobin acetaldehyde adducts. Most notably are assays for CDT that have been commercialized and are being used in many laboratories under routine conditions. This paper provides insight into the development, specifications, and use of the currently known CE-based assays suitable to detect alcohol markers. The achievements reached so far indicate that CE is an attractive technology for monitoring alcohol markers. This is particularly seen with the CDT assays that do not require an elaborate sample pretreatment and thus could be fully automated for high-throughput analyses on multicapillary instruments.

Determination of genetic transferrin variants in human serum by high-resolution capillary zone electrophoresis(†)

High-resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate-deficient transferrin in human serum. The assay comprises mixing of human serum with a Fe(III) -containing solution prior to analysis of the iron-saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, one BD and three CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high-resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate-deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.

Transferrin immunoextraction for determination of carbohydrate-deficient transferrin in human serum by capillary zone electrophoresis

CZE-based assays for carbohydrate-deficient transferrin (CDT) in which serum is mixed with an Fe(III) ion-containing solution prior to analysis are effective approaches for the determination of CDT in patient samples. Sera of patients with progressed diseases, however, are prone to interferences comigrating with transferrin (Tf) that prevent the proper determination of CDT by CZE in these samples. The need of a simple and economic approach to immunoextract Tf from human serum prompted us to investigate the use of a laboratory-made anti-Tf spin column containing polyclonal rabbit anti-human Tf antibodies linked to Sepharose 4 Fast Flow beads. This article reports extraction column manufacturing and column characterization with sera having normal and elevated CDT levels. The developed procedure was applied to a number of relevant hepatology and dialysis patient samples and could thereby be shown to represent an effective method for extraction and concentration of all Tf isoforms. Furthermore, lipemic sera were delipidated using a mixture of diisopropyl ether and butanol prior to immunoextraction. CDT could unambiguously be determined in all pretreated samples.

Therapeutic drug monitoring of cefepime with micellar electrokinetic capillary chromatography: Assay improvement, quality assurance, and impact on patient drug levels

The improvement and performance of a micellar electrokinetic capillary chromatography assay for cefepime in human serum and plasma with a 50 μm id fused-silica capillary elongated from 40 to 60 cm is reported. Sample preparation with dodecylsulfate protein precipitation at pH 4.5, the pH 9.1 separation medium and the applied voltage were as reported previously[16]. The change resulted in a significant lower current, higher resolution and increased detection time intervals. The performance of the assay with multi-level internal calibration was assessed with calibration and control samples. Quality assurance data of a two year period assessed under the new conditions demonstrated the robustness of the assay. In serum samples of patients who received both cefepime and sulfamethoxazole, cefepime could not be detected due to the inseparability of the two compounds. The presence of an interference can be recognized by an increased peak width (width > 0.2 min), the appearance of a shoulder or an unresolved double peak. The patient data gathered during a three year period reveal that introduction of therapeutic drug monitoring led to a 50% reduction of the median drug level. The data suggest that therapeutic drug monitoring can help to minimize the risk of major adverse reactions and to increase drug safety on an individual basis. This article is protected by copyright. All rights reserved.

Methylation of NR3C1 is related to maternal PTSD, parenting stress and maternal medial prefrontal cortical activity in response to child separation among mothers with histories of violence exposure.

Prior research has shown that mothers with Interpersonal violence-related posttraumatic stress disorder (IPV-PTSD) report greater difficulty in parenting their toddlers. Relative to their frequent early exposure to violence and maltreatment, these mothers display dysregulation of their hypothalamic pituitary adrenal axis (HPA-axis), characterized by hypocortisolism. Considering methylation of the promoter region of the glucocorticoid receptor gene NR3C1 as a marker for HPA-axis functioning, with less methylation likely being associated with less circulating cortisol, the present study tested the hypothesis that the degree of methylation of this gene would be negatively correlated with maternal IPV-PTSD severity and parenting stress, and positively correlated with medial prefrontal cortical (mPFC) activity in response to video-stimuli of stressful versus non-stressful mother-child interactions. Following a mental health assessment, 45 mothers and their children (ages 12-42 months) participated in a behavioral protocol involving free-play and laboratory stressors such as mother-child separation. Maternal DNA was extracted from saliva. Interactive behavior was rated on the CARE-Index. During subsequent fMRI scanning, mothers were shown films of free-play and separation drawn from this protocol. Maternal PTSD severity and parenting stress were negatively correlated with the mean percentage of methylation of NR3C1. Maternal mPFC activity in response to video-stimuli of mother-child separation versus play correlated positively to NR3C1 methylation, and negatively to maternal IPV-PTSD and parenting stress. Among interactive behavior variables, child cooperativeness in play was positively correlated with NR3C1 methylation. Thus, the present study is the first published report to our knowledge, suggesting convergence of behavioral, epigenetic, and neuroimaging data that form a psychobiological signature of parenting-risk in the context of early life stress and PTSD.