Specificity of the cannabinoid metabolite and phencyclidine EMIT d.a.u. assays

An investigation to determine the specificity of the EMIT d.a.u. assays was conducted. Samples of drug-free urine from healthy volunteers were spiked individually with one of 162 drugs to a concentration of 1000 mg/L. These samples were analyzed with the EMIT d.a.u. assays for phencyclidine and cannabinoids. Although some of the assays yielded positive results at this concentration, negative results were obtained in all samples diluted to 100, 10, and 1 mg/L.     

Evaluation of phencyclidine by EMIT d.a.u. utilizing the ETS analyzer and a 25-ng/mL cutoff.

This study evaluates the usefulness of the EMIT d.a.u. phencyclidine assay using the Syva ETS instrument to reliably detect phencyclidine in urine specimens at 25 ng/mL and above. More than 1600 urine specimens were screened over a one-month period. Fifty three (53) specimens screened positive (25 of these were previously tested positive by EMIT and GC/MS at or above the 25-ng/ml level and resubmitted as unknowns). Of the 53 specimens, 52 were confirmed positive by GC/MS. Reanalysis by EMIT of the one sample that was confirmed negative by GC/MS yielded a negative result. The absorbance difference between a negative sample and a sample containing 25 ng/mL of phencyclidine averaged 53 absorbance units with a standard deviation of 7.7 units. The absorbance difference between a negative sample and a sample containing 75 ng/mL of phencyclidine averaged 105 absorbance units with a standard deviation of 6.8 units.     

Improved sensitivity of digoxin assay by modification of the EMIT 2000 method

A modified EMIT 2000 digoxin assay was developed on the Cobas Mira plus analyzer for the determination of very low plasma concentrations of the drug. The major modifications were a higher plasma volume withdrawn during the analysis step and calibration curves constructed in the range 0-2 ng/ml using calibrators made up with biological matrix. Assays were controlled with an internal, four-level quality control (targets: 0.15; 0.60; 1.70; 2.70 ng/mL). The within-day and day-to-day mean observed values +/- SD (n = 10) of these quality controls were 0.14 +/- 0.02 and 0.15 +/- 0.02 ng/mL; 0.57 +/- 0.01 and 0.64 +/- 0.03 ng/mL; 1.55 +/- 0.06 and 1.62 +/- 0.04 ng/mL, 2.82 +/- 0.09 and 2.82 +/- 0.12 ng/mL, respectively. The detection and the quantification limits were 0.02 and 0.08 ng/mL, respectively. No significant difference was observed between digoxin plasma concentrations measured by the original and the modified EMIT 2000 digoxin assay in 25 plasma specimens, ranging from 0.4 to 3.0 ng/mL, from patients receiving the drug. This modified digoxin EMIT 2000 assay was subsequently used to study digoxin pharmacokinetics after each of 18 healthy volunteers was administered a single 0.5 mg oral dose. The pharmacokinetic parameters found in this study were in accordance with the literature in healthy subjects, using radioimmunoassay (RIA) for digoxin plasma concentration determinations. In conclusion, the lower limit of quantification of this modified EMIT 2000 digoxin assay is similar to that of RIA and can serve as a valuable screen for digoxin pharmacokinetic interactions studies.     

Clinical evaluation of the EMIT procainamide and N-acetylprocainamide assay.

Procainamide and its major metabolite, N-acetylprocainamide, were measured by the homogeneous enzyme immunoassay technique (EMIT). The reagents for the EMIT assays were supplied as a separate matched set for each assay. There is no cross-reactivity by procainamide in the assay for N-acetylprocainamide or by N-acetylprocainamide in the assay for procainamide. Within-day precision determined by replicate analysis of samples in the therapeutic range gave a coefficient of variation of less than 5% for each assay. The day-to-day coefficient of variation was less than 6% for each assay. Quantitative results obtained by the enzyme immunoassay on serum samples from patients receiving procainamide were compared with the results obtained by a high pressure liquid chromatography procedure. For the procainamide assay the correlation coefficient (r) was 0.983; for the N-acetylprocainamide assay the correlation coefficient was 0.981. There was no false positives or false negatives. The immunoassay requires 50 microliters of serum and the enzyme activity is measured in a spectrophotometer. An individual determination requires only 1 min to perform; therefore, the procedure can be used for either emergency or routine analysis.     

Comparison of an HPLC method with a RIA, EMIT and FIA method for the assay of serum gentamicin with extensive statistical evaluation

A high performance liquid chromatography (hplc) assay, a radioimmunoassay (ria), an enzyme immunoassay (emit) and a fluorescence immunoassay (fia) method for assaying serum gentamicin were compared in terms of accuracy and precision. Spiked human serum samples in the range of 0–11.6 mg.l^–1 gentamicin were used in all the tests.Regression analysis and analysis of variance were performed. The Performance Index (pi) was used to qualify the different methods. Other aspects of performance were also compared: simplicity, speed, cost, application in relation to workload.After extensive statistical evaluation thefia method gives the best results.     

Urinary screening for alprazolam, triazolam, and their metabolites with the EMIT d.a.u. benzodiazepine metabolite assay

Alprazolam (Xanax) and triazolam (Halcion) are relatively new triazolobenzodiazepines that are anxiolytic and hypnotic. This study assesses the reactivity of these drugs and their major metabolites in the EMIT d.a.u. benzodiazepine metabolite assay. Analytical standards of drugs and metabolites and urine specimens from patients receiving these drugs were analyzed by EMIT. Alprazolam and alpha-OH alprazolam gave an equivalent response to the EMIT low calibrator between 0.2 and 0.3 microgram/mL. Triazolam and alpha-OH triazolam were reactive between 0.3 and 0.5 microgram/mL. The assay was positive in 24 out of 27 random urine specimens from alprazolam-treated patients and in 8 out of 19 urine specimens from triazolam-treated patients. Positive urine results were confirmed by measuring the major urinary metabolites alpha-OH alprazolam and alpha-OH triazolam by HPLC. The study demonstrates that the EMIT assay can detect significant amounts of alprazolam and metabolites in the urine. The assay was negative in 58% of the specimens from individuals receiving triazolam, however.     

Modification of the EMIT tox benzodiazepine assay for screening of alprazolam in serum

Alprazolam (Xanax) is a relatively new triazolobenzodiazepine which is anxiolytic in man and is also prescribed in the treatment of panic attacks. The objective of this study was to optimize the reactivity of the EMIT tox serum benzodiazepine assay for toxicologic screening of alprazolam. The standard EMIT procedure was extensively modified so that therapeutic concentrations of alprazolam could be detected. Modifications for Methods A and B included a single dilution of specimens, increasing incubation temperature to 37 degrees C, dilution of Reagents A and B, and increasing specimen volume to 100 microL. Method B also included supplementing Reagent A with additional glucose-6-phosphate and beta-nicotinamide adenine dinucleotide. Using serum standards, the modified EMIT assays have detection limits of approximately 25 ng/mL (Method A) and 20 ng/mL (Method B). The patient specimens analyzed were considered positive by Method B and 24/28 positive by Method A. All 28 patient specimens tested by Method B were confirmed positive by gas chromatography/electron capture detection. The day-to-day precision of both methods was less than 2% CV with diluted EMIT calibrators (N = 6). In conclusion, the modified EMIT assay can be used to screen for alprazolam in serum at therapeutic concentrations.     

异硫氰酸苯甲酰酯稳定性指示性气相分析方法开发和验证

目的:建立异硫氰酸苯甲酰酯稳定性指示性气相分析方法.方法:气相色谱柱DB-1(30m×0.25mm×0.25μm);载气:氦气;检测器:氢焰离子化检测器;起始柱温为100℃,维持1.3min,每分钟15℃的速率升温至200℃,然后以每分钟5℃的速率升温至240℃,再以每分钟10℃的速率升温至300℃,维持6min;进样口温度:200℃;检测器温度:300℃;柱流量:1.0mUmin;进样量:1μL.结果:专属性、线性、定量限、检测限、精密度、溶液稳定性、准确度经验证,结果均良好.结论:本法可用于异硫氰酸苯甲酰酯的初步稳定性研究.     

Multidrug assay method for antimalarials.

A general separation strategy, involving solid-phase extraction followed by reversed-phase ion-pairing HPLC with an organic counter ion for a set of 11 widely used antimalarial drugs and metabolites has been developed. The basis underlying the separation has been explored and work, including quantitative data, has been carried out on illustrative separations which form the basis of novel quantitative assays of groups of antimalarials which are relevant to current prophylaxis and treatment of malaria.     

Development and validation of a capillary electrophoresis method for ximelagatran assay and related substance determination in drug substance and tablets.

The advantages of simplicity, selectivity, versatility and ease of use of free solution capillary electrophoresis (CE) present an orthogonal and complementary separation technique to the established methods of liquid chromatography (LC) for pharmaceutical analysis. This work presents the development and performance of a suitable CE method for ximelagatran (formerly H 376/95) assay and related substance determination in both drug substance and tablets. The method employed was a low pH phosphate buffer, to which acetonitrile and hydroxypropyl-beta-cyclodextrin were added, in order to facilitate the separation of ximelagatran and its related substances. An applied field of 350 V/cm was used and all compounds were resolved in approximately 20 min. Benzamidine hydrochloride was used as an internal standard in quantification. The data indicate that the performance of the validated method offers equivalent and complementary information, in terms of selectivity, sensitivity, accuracy, linearity and precision, to that of an established gradient LC method employed for similar purposes. Robustness of the method was investigated by experimental design and evaluated using multivariate calculations.     

Development and validation of a stability indicating HPLC assay method for cyclosporine in cyclosporine oral solution USP

The suitability of the United States Pharmacopeia (USP) assay method for analysing stressed samples of cyclosporine oral solution USP was evaluated for stability samples by analyzing cyclosporine oral solution after acid, alkali, hydrogen peroxide, heat and light treatment. Some of the degradants generated during stress testing, as well as dihydrocyclosporine A, which is a known degradant of cyclosporine A, were not adequately resolved from the cyclosporine peak and mobile phase adjustments did not improve the resolution. In addition, isocyclosporine A, another known degradant of cyclosporine, could not be quantitated as it was eluting too early with the system peaks. Therefore, a binary gradient, reverse phase, stability indicating, HPLC method for the assay of cyclosporine in cyclosporine oral solution USP has been developed and validated. Analysis of degraded samples showed that the cyclosporine A eluted as a spectrally pure peak resolved from its degradation products.     

Correlation of the "EMIT" with a gas-liquid chromatographic method for determination of antiepileptic drugs in plasma.

We report a study of the comparison of antiepileptic drugs (phenobarbital, phenytoin, primidone, and carbamazepine) by the Enzyme Multiplied Immunoassay Technique "EMIT" and the gas chromatography (GLC) method. Overall, reasonable correlations were observed for all of the above-mentioned assays by both methods in the majority of samples included in this study. Our observations for statistical and clinical differences at various levels of phenobarbital and phenytoin are discussed. A suggestion is provided in order to avoid a major discrepancy (approximately 30% higher values by EMIT vs GLC) in results observed from the "Bottom of the Bottle Effect" for EMIT reagent.     

Development and validation of a reverse-phase liquid chromatographic method for the assay of lidocaine hydrochloride in alginate-Gantrez microspheres

A simple, fast and reliable reverse-phase high-performance liquid chromatographic (HPLC) method was developed for the assay of lidocaine hydrochloride (LH) in Gantrez-alginate microspheres. Separation was achieved in a LiChrospher C18 column, using a mobile phase consisting of acetonitrile:ammonium acetate (0.0257 M) adjusted to pH 4.85 with acetic acid, in the ratio 70:30 (v/v) and a flow rate of 0.6 mL/min. The detection was made with a diode array detector measuring at the maximum for the compound. The validation study demonstrated that the method was precise, accurate and linear over the concentration range of analysis with a limit of detection of 0.001 mg/mL. The limit of quantification was 0.002 mg/mL. Linear regression analysis in the range of 0.8-2.4 mg/mL gave correlation coefficients higher than 0.995. The method developed was applied to the analysis of lidocaine in microsphere samples in order to evaluate in next papers, the encapsulation efficiency of different formulations.     

Development and validation of a stability-indicating RP-HPLC method for assay of betamethasone and estimation of its related compounds

Betamethasone (9α-fluoro-16β-methylprednisolone) is one of the members of the corticosteriod family of active pharmaceutical ingredient (API), which is widely used as an anti-inflammatory agent and also as a starting material to manufacture various esters of betamethasone. A stability-indicating reverse-phase high performance liquid chromatography (RP-HPLC) method has been developed and validated which can separate and accurately quantitate low levels of 26 betamethasone related compounds. The stability-indicating capability of the method was demonstrated through adequate separation of all potential betamethasone related compounds from betamethasone and also from each other that are present in aged and stress degraded betamethasone stability samples. Chromatographic separation of betamethasone and its related compounds was achieved by using a gradient elution at a flow rate of 1.0 mL/min on a ACE 3 C18 column (150 mm × 4.6 mm, 3 μm particle size, 100 Å pore size) at 40 °C. Mobile phase A of the gradient was 0.1% methanesulfonic acid in aqueous solution and mobile phase B was a mixture of tert-butanol and 1,4-dioxane (7:93, v/v). UV detection at 254 nm was employed to monitor the analytes. For betamethasone 21-aldehyde, the QL and DL were 0.02% and 0.01% respectively. For betamethasone and the rest of the betamethasone related compounds, the QL and DL were 0.05% and 0.02%. The precision of betamethasone assay is 0.6% and the accuracy of betamethasone assay ranged from 98.1% to 99.9%.     

Development and validation of a reversed-phase liquid chromatographic method for the assay of lidocaine in aqueous humour samples

A simple, fast and reliable reversed-phase liquid chromatographic method was developed for the assay of lidocaine in human aqueous humour samples. The samples were analysed without any preliminary treatment on a C8 column with UV detection at 225 nm. The mobile phase consisted of methanol/sodium dihydrogen phosphate (30 mM) containing sodium pentansulphonate (10 mM) adjusted to pH 2.5 with phosphoric acid (50:50 v/v). Validation of the method showed it to be precise, accurate and linear over the concentration range of analysis with a limit of detection of 0.2 μg ml⁻¹. The limit of quantitation was 2.5 μg ml⁻¹ with a relative standard deviation of 2.5%. Linear regression analysis in the range 2.5–60 μg ml⁻¹ gave correlation coefficients higher than 0.999. No interference from three commonly co-administered drugs was observed. The method developed was applied to the analysis of lidocaine in aqueous humour samples in order to evaluate and compare the efficacy of two different forms of administration of lidocaine for topical anaesthesia in cataract surgery.     

Development and validation of a new assay for assessing clot integrity

IntroductionResearch and routine laboratory assessment of clot integrity can be time consuming, expensive, and cannot be batched as it is generally performed in real time. To address these issues, we developed and validated a micro-titre based assay to quantify thrombogenesis and fibrinolysis, the purpose being to assess patients at risk of cardiovascular events by virtue of hypercoagulability. In further validation, thrombogenesis results were compared to similar indices from the thrombelastograph (TEG).MethodsOur assay determines three indices of thrombogenesis (lag time to the start of thrombus formation (LT), rate of clot formation (RCF), and maximum clot density (MCD)) and two of fibrinolysis (rate of clot dissolution (RCD) and time for 50% of the clot to lyse (T50)). Plasma was tested fresh and again after being frozen at − 70 °C. Some samples were tested immediately, others after being left at room temperature for up to 24 h.ResultsThe intra-assay coefficients of variation (CVs) of the three thrombogenesis measures (LT, RCF, MCD) and two fibrinolysis measures (RCD, T50) varied between 2.7 and 12.0% in fresh plasma and between 1.3% and 10.8% in frozen plasma respectively. Similarly, the inter-assay coefficients of variation of the thrombogenesis and fibrinolysis measures were 4.9–10.8% in fresh plasma and 2.2–6.5% in frozen plasma respectively. TEG assays intra- and inter assay CVs were around 25%. There were no significant differences in all plate assay indices up to 6 h at room temperature. Certain plate assay thrombogenesis data were comparable to TEG indices after analysis by Pearson's correlation. The reagent processing cost per sample is £15 for TEG and £2 for the plate assays.ConclusionOur micro-titre based assay assessing plasma thrombogenesis and fibrinolysis has good intra- and inter-assay CVs, can assess plasma up to 6 h after venepuncture, is more efficient (in terms of throughput) and is more economical than that of the TEG.     

Semiquantitation of cannabinoid immunoassays? A reexamination of the EMIT 20-ng/mL assay.

Concerns regarding the ability to semiquantitate drugs-of-abuse urine immunoassay results, particularly THC-COOH, prompted us to reexamine EMIT results. The Syva EMIT d.a.u. cannabinoid 20-ng/mL assay was performed on the Cobas Bio centrifugal analyzer, and positive samples were confirmed by Toxi-Lab or GC/MS. Of 39 specimens tested, 17 were confirmed positive. However, four specimens were not accurately semiquantitated by EMIT. These four had very high levels (greater than 100 ng/mL) of the primary metabolite but yielded EMIT results between the low and medium calibrator (20-75 ng/mL). Linearly studies confirmed that the absorbance changes with the EMIT immunoassay plateau near the medium calibrator. In addition, we obtained false positive EMIT results due to sample carryover when samples containing 500 ng/mL (or greater) of THC-COOH preceded a negative specimen. Significant carryover was not observed when concentrations up to 1,300 ng/mL were used with the 100-ng/mL immunoassay, and this effect may be explained by the higher cutoff used. Because EMIT cannabinoid results on the Cobas Bio analyzer can be affected by carryover and a hook effect, they should not be reported even semiquantitatively.