Intra-day Variabilities Research Articles

Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair.

Flunitrazepam (Rohypnol) is a benzodiazepine used in the treatment of insomnia as a sedative hypnotic and as preanesthetic medication in European countries and Mexico. Although it has no medicinal purpose in the United States, the occurrence of its abuse is increasing. Sexual abuse of both men and women while under the influence of so-called "date-rape" drugs has been the focus of many investigations. Reported date-rape drugs include flunitrazepam (FN), clonazepam, diazepam, oxazepam, gamma-hydroxybutyrate, and many others. FN has been banned in the United States because of its alleged use in such situations. Unfortunately, the detection of FN or its metabolites 7-aminoflunitrazepam (7-AFN) and desmethylflunitrazepam in a single specimen such as urine or blood is difficult in criminal situations because of the likelihood of single-dose ingestion and the length of time since the alleged incident. Hair provides a solution to the second of these problems in that drugs tend to incorporate into hair and remain there for longer periods of time than either urine or blood. There are various techniques for the detection of FN in plasma, blood, and urine, but little work has been done with hair. Hair collection is a virtually noninvasive procedure that can supply information on drug use for several months preceding collection. The objective of this paper was to determine if a commercially available micro-plate enzyme immunoassay system was sufficiently sensitive for the routine screening of 7-AFN in hair by the development of extraction procedures and optimization of the immunoassay kit. Further, this study used the same solid-phase extraction to isolate FN and its major metabolite, 7-AFN, and gas chromatography-mass spectrometry with negative ion chemical ionization for confirmation. Two seven-point standard curves were established ranging from 0.5 pg/mg to 100 pg/mg for 7-AFN and 2.5 pg/mg to 200 pg/mg for FN with respective deuterated internal standards. A replicate analysis of controls was performed to establish inter- and intraday variabilities. Two suicide cases along with one alleged date-rape case and one case of an emergency room patient whose blood screened positive for benzodiazepines were analyzed. All the hair specimens screened positive for benzodiazepines using micro-plate enzyme immunoassay. Two cases, including the date-rape case, were negative for FN and 7-AFN, and two postmortem hair samples were confirmed positive for FN and its metabolite.

Long-Term Stability of 8-Methoxypsoralen in Ointments for Topical PUVA Therapy (‘Cream-PUVA’)

8-Methoxypsoralen (8-MOP) is an established photochemotherapeutic agent for PUVA therapy. Recently, a so-called ‘cream-PUVA’ modality was introduced into therapy of psoriasis and other dermatoses. Little is known, however, about the stability of 8-MOP in ointments used for the topical application of this compound. Therefore, we investigated the long-term stability of 8-MOP in three different ointments, Unguentum Cordes^TM, Cold Cream Naturel^TM and a water-containing gel on the basis of Carbopol 940. All three ointments were prepared with 8-MOP concentrations of 0.05 and 0.005%, and stored over 12 weeks at room temperature (19–20°C) and at 5°C. 8-MOP concentrations were measured at days 1, 8, 15, 29, 57 and 88 after preparation by thin-layer chromatography (TLC). The ointments were dissolved in an organic solvent, 10 µl were transferred onto the TLC plate and the chromatograms were developed first in toluene and then in toluene/ethyl acetate 2:1 v/v to resolve 8-MOP from the ointment constituents. The peak heights of 8-MOP were used for quantitation. The intraday variabilities are <3% for Unguentum Cordes and Cold Cream Naturel and <6% for the Carbopol 940 gel. The interday variabilities were <6.3% in all cases. In Unguentum Cordes and Cold Cream Naturel the concentrations of 8-MOP remain stable, but in Unguentum Cordes the emulsion began to break up after 8 weeks. In the Carbopol gel, only about 40% of the nominal concentrations of 8-MOP were found and they decrease significantly at storage at 5°C. We conclude that the Carbopol gel seems to be unsuitable for PUVA therapy, whereas Cold Cream Naturel shows the best results.

Analysis of doxorubicin in cell culture media and human plasma using solid phase extraction and HPLC

Doxorubicin is an antineoplastic antibiotic isolated fromStreptomyces peucetius var.cesius clinically used in the treatment of tumors such as lung or breast, Hodgkin's disease and various types of leukemias. The main goal of this study was to develop a simple and sensitive HPLC method with fluorescence detection for the quantitation of doxorubicin in cell culture media collected during an in vitro studies and in human plasma. Solid phase extraction (C2 silica) was applied. The experiment established five-point standard curve (1 ng mL−1 to 100 ng mL−1). The standard curves prepared in blank cell tissue media were linear over the range of doxorubicin assayed and had a mean correlation coefficient of 0.9973±9.43×10−4 and slope 0.02545±1.85×10−3. The standard curves prepared in human plasma were linear and had mean correlation coefficient of 0.997 and slope 0.01885±5.19×10−4. The limit of quantitation for doxorubicin in both specimens was arbitrarily established to be 1 ng mL−1. Intra-day variabilities were determined using 3–4 replicates of control solutions of doxorubicin (3 ng mL−1 and 30 ng mL−1) in blank plasma and cell culture media. Inter-day variabilities were determined over a four day period analyzing replicates of controls. All precision and accuracy values fell within the acceptable range.

Quantification of fluoxetine and norfluoxetine serum levels by reversed-phase high-performance liquid chromatography with ultraviolet detection

A rapid and sensitive high-performance liquid chromatography assay method was developed to determine serum fluoxetine and norfluoxetine levels by single extraction of 0.1 ml of serum with sodium hydroxide. The mobile phase (55% acetonitrile–45% distilled water containing 10 m M aqueous triethylamine) was used to separate fluoxetine and norfluoxetine (25–1000 ng/ml, using clomipramine as the internal standard) by ultraviolet detection at 226 nm. The inter- and intra-day variabilities of fluoxetine and norfluoxetine were 13–18%, and the recoveries of both drugs exceeded 89%. This assay method was applied to a pharmacokinetic disposition study of fluoxetine in mice.

An HPLC/RIA method for dynorphin A1-13 and its main metabolites in human blood

A selective HPLC/RIA procedure for the determination of dynorphin A1-13 (Dyn A1-13) and its major metabolites in human blood was developed. In order to block peptidase activity, blood samples were transferred into an aliquot of a blocking solution (5% aqueous ZnSO 4 solution-acetonitrile-methanol; 5:3:2, v/v/v). After solid phase extraction, reconstituted aliquots were injected into an isocratic reversed phase HPLC system to separate Dyn A1-13 from its main metabolites (Dyn A2-13, Dyn A1-12 and Dyn A2-12). The isolated and concentrated HPLC-fractions were assayed by RIA using a commercially available antiserum. Intra-day variabilities for quality controls (0.07, 0.25, and 1 ng ml −1) of Dyn A1-13, A2-13, A1-12, A2-12 were between 9 and 41%. Accuracy was between 86 and 132%. Inter-day variability for single quality controls analyzed on five days for Dyn A1-13, A2-13, A1-12, A2-12 was between 4 and 49% for 0.07, 0.25 and 1 ng ml −1 samples, respectively. Accuracy was between 72 and 129%. Five different batches of control blood showed blood levels no different from zero. Considering the complexity of the assay, the method is selective, accurate and reproducible with a limit of detection of 0.07 ng ml −1 for Dyn A1-13, Dyn A2-13, Dyn A1-12 and 0.21 ng ml −1 for Dyn A2-12. The assay was applied to the determination of Dyn A1-13 and its metabolites in blood samples of 2 subjects receiving i.v. infusions of 250 μg or 1000 μg kg −1 Dyn A1-13 over 10 min.

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection.

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. alpha-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 microg/ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 microg/ml were <3% (n=5).

Fluorometric High Performance Liquid Chromatography for Quantitation of Naproxen in Serum

Abstract A simple, sensitive and reliable HPLC method for the determination of naproxen in serum is presented. Samples were prepared by a protein precipitation method using acetonitrile. A reversed-phase C-18 column was used with a mobile phase consisting of 47% acetonitrile and 53% pH 2.5 buffer solution. A fluorescence detection with excitation wavelength of 230 nm and emission wavelength of 370 nm provided high sensitivity and specificity with no interference from normal serum constituents. The limit of quantitation was 2.0 ng/ml with a CV of 10.6%. The standard plots were highly linear (r>0.999) over the range of 10.0–200.0 ng/ml. The average recovery was 97% and the CVs of inter- and intra-day variabilities were 2.0% and 1.2%, respectively, for the entire calibration range. The method was used for the measurement of the time course of naproxen in dog serum after administration of a topical gel.

Selective stability-indicating high-performance liquid chromatographic assay for recombinant human regular insulin.

This report presents a selective HPLC assay capable of separating recombinant human regular insulin from insulin decomposition and transformation products. The assay utilizes an isocratic delivery of mobile phase, a C18 peptide column, UV detection and is performed at ambient temperature. The standard curve ranges from 0.2 to 2.5 U/ml. The inter-day and intra-day variabilities are less than 7 and 5%, respectively, at the concentrations studied. The accuracy and precision are within 5% over the range of the standard curve.

Simultaneous analysis of diphenylmethoxyacetic acid, a metabolite of diphenhydramine, and its deuterium-labeled stable isotope analog in ovine plasma and urine.

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([2H10]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography-electron-impact mass spectrometry (GC-EI-MS) analysis of DPMA and [2H10]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-microliter aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [2H10]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [2H10]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [2H10]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [2H10]DPMA in biological samples obtained from sheep (e.g. plasma and urine).

A selective LC/RIA for dexamethasone and its prodrug dexamethasone-21-isonicotinate in biological fluids

A combined LC/RIA procedure is described for the selective determination of dexamethasone (DEX) and its prodrug dexamethasone-21-isonicotinate (DIN) in plasma. The low affinity of the employed dexamethasone antiserum for DIN (cross-reactivity less than 0.5%) allowed the direct determination of DEX in plasma extracts. For the determination of DIN, both substances of interest were separated by LC, the DIN containing fraction was collected, hydrolysed and the generated DEX was consequently assayed by radioimmunoassay. The assay detection limits were 0.1 ng ml-1 for DEX and 0.75 ng ml-1 for DIN. For both substances, inter- and intra-day variabilities (RSDs) were 6 and 12%, respectively.