A Gas Chromatographic Method for the Determination of Amitriptyline and Nortriptyline in Human Serum

Abstract: Amitriptyline (AMT) and nortriptyline (NT) were extracted from serum with hexane at pH 9. An internal standard was added and extracted with the drugs. During the concentration of the hexane phase nortriptyline was acetylated to improve separation. One fifth of the final hexane phase was injected into a gas chromatograph equipped with a detector, which was selectively sensitive towards nitrogen. The lower limits of detection were 5 ng/ml for AMT and 10–15 ng/ml for NT. A constant relative standard deviation of 7.6% was found in the concentration interval used for the standard curves. No interference appeared from AMT metabolites and some benzoediazepines. The method has been used for the estimation of AMT after a single dose of this drug to two human volunteers and for the estimation of AMT and NT after repeated administration of amitriptyline to patients. After single dose administration NT was not detectable, while the NT concentration was of the same order as the AMT concentration after repeated administration. A great individual variation in the concentrations of the two drugs was seen after repeated administration.     

GC-MS quantitation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in blood

A method is described for the simultaneous analysis of seven opiates, codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone, in blood samples by gas chromatography-mass spectrometry (GC-MS). One milliliter of blood is combined with an internal standard mixture containing 200 ng of each of the seven deuterated opiates. Two milliliters of acetonitrile is added to precipitate the proteins and cellular material. After centrifugation, the clear supernatant is removed, and the acetonitrile is evaporated. The remaining aqueous portion is adjusted to pH 9 with sodium bicarbonate buffer, and the drugs are extracted into chloroform/ trifluoroethanol (10:1). The organic extractant is transferred and dried under nitrogen. The residue is reconstituted in dilute hydrochloric acid and washed consecutively with hexane and chloroform. The purified aqueous portion is adjusted to pH 9 with bicarbonate buffer, and the drugs are again extracted into chloroform/trifluoroethanol (10:1). The organic portion is removed from the aqueous fraction and dried under nitrogen. The residue is consecutively derivatized with methoxyamine and propionic anhydride using pyridine as a catalyst. The ketone groups on hydrocodone, hydromorphone, oxycodone, and oxymorphone are converted to methoximes. Hydroxyl groups present at the O(3) and O(6) positions of codeine, morphine, 6-acetylmorphine, hydromorphone, and oxymorphone are converted to their respective propionyl esters. After a post-derivatization purification step, the extracts are analyzed by full scan GC-MS using electron impact ionization. The method is linear to at least 2000 ng/mL. Day-to-day precision (N = 15) at 500 ng/mL and 75 ng/mL were less than 10% for all seven targeted opiates. Extraction efficiencies at these two concentrations ranged from 50% to 68%. For each opiate, the limit of quantitation was 10 ng/mL, and the limit of detection was 2 ng/mL.     

荧光离子对测定叔胺类药物的研究

本文研究了用荧光试剂9,10-二甲氧基蒽-2-磺酸钠(MAS)与叔胺类药物作用生成离子对,经溶剂萃取,测定叔胺的方法。对影响生成荧光离子对的主要因素,如萃取介质pH的影响,试剂用量和合适有机溶剂的选择等进行了研究。用本法测定了9,10-二甲氧基蒽-2-磺酸钠的量子产率和六种叔胺类药物低浓度范围(10^-8～10^-7M)的线性区间、最低检出限以及方法的精密度。本法操作简便,灵敏度高,最低检出限为1 ng/ml,变异系数小于5%。同紫外分光光度法和酸性色素离子对法相比,灵敏度分别高出1000和100倍。     

A simple high-performance liquid chromatography method for the quantitation of tricyclic antidepressant drugs in human plasma or serum

OBJECTIVE: A simple reversed-phase high performance liquid chromatography method with a variable wavelength detection has been developed for determining the commonly used tricyclic antidepressant drugs in plasma. METHODS: The assay procedure involves a liquid-liquid extraction with an initial extraction into hexane:ethyl acetate after basification of the homogenate. The column used was a Supelco Hypersil 5 microm, 150 x 3.2 mm. The mobile phase was acetonitrile, methanol, and phosphate buffer (0.01M, pH 7.4) at 12:3:5 ratio (v/v/v). RESULTS: The recoveries ranged from 89 to 108% and the day-to-day precision was 1.1 to 13% CV depending on the compound. The method is sensitive to 15 ng/mL and linear to 400 ng/mL with a 25 microL injection. CONCLUSION: This method is simple, sensitive, precise, inexpensive, and is currently used in our laboratory for therapeutic monitoring of tricyclics. The data generated by this method were within the range outlined by the College of American Pathologists.     

A sensitive gas chromatographic assay for the determination of serum viloxazine concentration using a nitrogen-phosphorus-selective detector

A gas-liquid chromatographic procedure for measuring the serum levels of the antidepressant viloxazine is described. The drug and the internal standard [imipramine (IMI)] are extracted from 1 ml serum. The method involves a three-step extraction, derivatization of viloxazine with acetic anhydride, and injection into a gas chromatograph equipped with a nitrogen-phosphorus-selective detector. The retention times for IMI and viloxazine were 4.7 and 6.1 min, respectively. The standard curves were linear over the 100- to 2,000-ng/ml range. The recovery averaged 64.5% and the lowest detection limit was 80 ng/ml. The within-run and day-to-day coefficients of variations were 11.9 and 12.5%, respectively, at 250 ng/ml, and 8.9 and 9.2%, respectively, at 1,500 ng/ml. The method is adequate both for single-dose pharmacokinetic studies and for monitoring serum viloxazine levels in chronically treated patients.     

Determination of paroxetine in geriatric depression by high-performance liquid chromatography.

An HPLC method was developed for the determination of paroxetine in depressed geriatric patients. This method includes only one-step extraction of paroxetine from 1 ml of plasma with hexane:isoamyl alcohol (99:1, v/v). Separation was obtained using an RP select B column (250 mm x 4 mm, 5 microm) under isocratic conditions with UV detection (lambda=205 nm). The mobile phase was 0.05 mol/l sodium phosphate buffer, pH 5.0, and acetonitrile (50:50, v/v). The intra- and inter-assay accuracy and precision, determined as relative error and relative standard deviation, respectively, were less than 10%. The lower limit of quantification, with relative error and relative standard deviation of less than 10%, was 5 ng/ml. Linearity was assessed in the 5-1250 ng/ml range and recovery was 80%. Neither endogenous compounds nor other drugs were found to interfere. The assay showed high specificity, even when patients were found to be on multiple medications. In conclusion, the present method, when compared to others reported in the literature, has advantages such as simplicity, selectivity and sensitivity as a routine procedure and can be applied to the therapeutic monitoring of depressed geriatric patients.     

Determination of the concentration of diallyl trisulfide in rat whole blood using gas chromatography with electron-capture detection and identification of its major metabolite with gas chromatography mass spectrometry

A simple, rapid, and sensitive procedure has been developed using gas chromatography with electron-capture detection to measure diallyl trisulfide levels in rat blood. Blood samples were acidified, and the analyte was extracted with hexane, and then degradation was stopped with acetonitrile before gas chromatographic separation. Two calibration curves were linear over the range of 10-500 ng/ml and 0.2-20 microg/ml, with typical r values of 0.9986 and 0.9993, respectively. The structure of its major metabolite was confirmed using combined gas chromatography-mass spectrometry. The limit of detection was less than 10 ng/ml, and the assay was highly reproducible, giving peaks with excellent chromatographic properties. The method is suitable for pharmacokinetic and therapeutic purposes.     

A sensitive thin-layer chromatographic procedure for the detection of urinary 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid

A sensitive high-performance thin-layer chromatographic (HPTLC) procedure for the detection of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), the major urinary metabolite of delta 9-tetrahydrocannabinol (THC), is described. After alkaline hydrolysis, the THC-COOH is extracted from acidified urine into hexane. The hexane is evaporated and the residue spotted onto an HPTLC plate. Chromatography time is 11 min with heptane/butanol/acetic acid (90:9:1) as mobile phase. The cannabinoids are visualized by sequentially dipping the thin-layer plate in diethylamine then in 0.1% Fast Blue BB. The limit of detection for THC-COOH is 5 ng/mL when 2 mL of urine is used. Extraction efficiencies averaged 71% over the 10-400 ng/mL range. No drugs, drug metabolites, or endogenous urinary substances interfere with the procedure.     

Development and Validation of a RP-HPLC Method for Determination of Cyclosporine in Capsule

A simple, specific and accurate reverse phase high performance liquid chromatographic method was developed for the determination of cyclosporine in capsule dosage form. XTerra C18 column was used as stationary phase with mobile phase acetonitrile in combination with 0.1% trifluoro acetic acid buffer and pH is adjusted to 1.4. Method was developed in an isocratic run of 20% trifluoro acetic acid with 80% acetonitrile for 10 min, at flow rate of 1 ml/min. Effluents were monitored at 210 nm. Retention time of cyclosporine was 3.855 min. The method was validated for specificity, linearity, accuracy, precision, limit of quantification, limit of detection, robustness and solution stability. Limit of quantification and limit of detection of cyclosporine was found to be 100 ng/ml and 200 ng/ml. Recovery was found to be in the range of 98.08-101.55%. The proposed method was successfully applied for the quantitative determination of cyclosporine in a capsule dosage form.     

Miniaturized hollow fibre assisted liquid‐phase microextraction and gas chromatography for determination of trace concentration of sufentanil and alfentanil in biological samples

A simple and highly sensitive method that involves miniaturized hollow fibre assisted liquid‐phase microextraction with gas chromatography‐flame ionization detector was developed for the determination of trace concentration of sufentanil and alfentanil in biological samples. These drugs were extracted from 5 ml of aqueous solution with pH 10.0 into an organic extracting solvent (1‐octanol) impregnated in the pores and lumen of a hollow fibre. After extraction for a prescribed time, 2.0 µl of the extraction solvent was injected directly in to the GC injection port. Under the optimized conditions, (1‐octanol as extracting solvent, stirring rate of 700 rpm, 15% (w/v) salt addition, pH 10.0 and 25 min sampling time at 50 °C) large enrichment factors of 535 and 420 were achieved for sufentanil and alfentanil, respectively. Dynamic linear ranges were in the range of 0.05 to 500 ng/ml for sufentanil and 0.1 to 500 ng/ml for alfentanil. Limits of detection 0.01 and 0.02 ng/ml were obtained for sufentanil and alfentanil, respectively. The percent relative intra‐day and inter‐day standard deviations were found to be less than 8.4% (n = 5). Finally, this method was successfully applied for the separation, preconcentration and determination of trace concentration of sufentanil and alfentanil in plasma and urine samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Liquid chromatographic determination of reduced haloperidol and haloperidol concentrations in packed red blood cells from humans

Haloperidol (H) is a neuroleptic drug that has one known biologically active metabolite, reduced haloperidol (RH). A liquid chromatographic method is described for the determination of both compounds in human red blood cell (RBC) samples. The drugs were extracted into hexane at high pH and back-extracted into 0.1M HCl. The acid solution was then analyzed by reversed-phase chromatography under the following conditions: column was ultrasphere ODS; eluant was acetonitrile:0.085M phosphate buffer (30:70), final pH was 3.5, flow rate was 2 mL/min; detection was by light absorption at 246 nm for H and 220 nm for RH. The minimum limits of quantitation for H and RH were 0.25 and 0.1 ng/mL of packed RBC respectively. For six selected patients on 10 or 20 mg per day of oral haloperidol the RBC to plasma concentration ratios for RH and H were 2.20 +/- 0.9 (SD) and 0.81 +/- 0.26, respectively. The data indicate that RH is more concentrated in RBC than in plasma.     

Simultaneous determination of aspirin and isosorbide 5-mononitrate in formulation by reversed phase high pressure liquid chromatography

A simple, precise and rapid reversed phase HPLC method was developed for the simultaneous estimation of aspirin (AS) and isosorbide 5-mononitrate (ISM) in combined formulation. The method was carried out on a Thermo Quest C18 column using a mixture of water:methanol (water pH adjusted to 3.4 using dilute orthophosphoric acid) and detection was carried out at 215 nm using chlorzoxazone as internal standard. Both the drugs showed linearity in the range of 2–10 μg/ml and limits of quantification was found to be 4 and 40 ng/ml for AS and ISM, respectively.     

Simultaneous measurement of bupivacaine, etidocaine, lidocaine, meperidine, mepivacaine, and methadone

A gas-liquid chromatographic method for the simultaneous measurement of bupivacaine, etidocaine, lidocaine, meperidine, mepivacaine, and methadone in serum is described. The drugs and the internal standard, prilocaine, are extracted from 1 ml of serum. The procedure involves a two-step extraction and injection of the extract into a gas chromatograph equipped with a 10-ft OV-11 glass column and a nitrogen-phosphorus detector. The temperature gradient program results in a run time of 16 min and retention times for meperidine, prilocaine (internal standard), lidocaine, etidocaine, mepivacaine, methadone, and bupivacaine of 3.8, 5.4, 6.0, 8.7, 11.0, 11.7, and 14.8 min, respectively. Standard curves for all drugs were linear over the 80 to 2,000-ng/ml range and recovery of all components averaged 97 +/- 2% with the lowest detection limit of 10 ng/ml for all drugs except meperidine and methadone, which were 20 ng/ml. The within-day coefficients of variation ranged from 12 to 8% at 500 ng/ml. The day-to-day coefficients of variation of the slope and intercept values ranged from 2 to 0% and 130 to 3%, respectively. Response factors of the nitrogen-specific collector varied with the drug analyzed and resulted in peak area variation at constant offset and attenuation of 30%. This method is intended and adequate for therapeutic monitoring of chronically treated pain patients who are being given various combinations of local anesthetic and/or narcotic agents.     

Determination of Codeine in Plasma by High-Performance Liquid Chromatography

An automated, sensitive, and selective reverse-phase high-performance liquid chromatographic assay has been developed to measure codeine in plasma. The analysis requires only 1 ml plasma and is accomplished by detection of the fluorescence of codeine following extraction and concentration. The method is simple and rapid, involving a one-step extraction of codeine from alkalinized (pH 10.0) plasma into an organic layer of hexane/dichloromethane, 2/1. The organic layer was evaporated under nitrogen and the residue reconstituted with the mobile phase. The samples were chromatographed on a reverse-phase C-18 column using a mobile phase of acetonitrile–phosphate buffer, 80/20 (pH 5.80). The codeine and internal standard, N-allylnorcodeine, peaks were detected using a fluorescence detector. The retention times were 8.6 min for the internal standard and 11.3 min for codeine. Standard curves were linear from 10 to 250 ng/ml. The assay was validated by direct comparison with a gas chromatographic procedure that employed nitrogen–phosphorus detection. The assay has been employed for the analysis of several codeine studies using human, dog, and rat plasma.     

Evaluation of a multi-class, multi-residue liquid chromatography-tandem mass spectrometry method for analysis of 120 veterinary drugs in bovine kidney

Traditionally, regulatory monitoring of veterinary drug residues in food animal tissues involves the use of several single-class methods to cover a wide analytical scope. Multi-class, multi-residue methods (MMMs) of analysis tend to provide greater overall laboratory efficiency than the use of multiple methods, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of targeted drug analytes usually provides exceptional performance even for complicated sample extracts. In this work, an LC-MS/MS method was optimized and validated in a test of 120 drug analytes from 11 different classes in bovine kidney. The method used 10 ml of 4/1 acetonitrile/water for extraction of 2 g samples and cleanup with hexane partitioning. Quantitative and qualitative performance was assessed for the analytes at fortification levels of 10, 50, 100, and 200 ng/g. With the method, 66 drugs gave 70-120% recovery with ≤ 20% RSD at all levels over the course of 3 days. At the 200 ng/g level, 89 drugs met these same standards. Limits of detection were ≤ 10 ng/g for 109 of the analytes in the kidney matrix in validation experiments. Qualitatively, MS/MS identification criteria were set that ion ratios occur within ± 10% (absolute value) from those of the analyte reference standards. At the 10 ng/g level, 57% of the drugs met the identification criteria, which improved to 84% at the 200 ng/g level. The method serves as an efficient and useful additional option among the current monitoring methods available.     

Determination of bisnafide,a novel bis-naphthalimide anticancer agent,in human plasma by high-performance liquid chromatography with UV detection

A simple, specific, and sensitive high-performance liquid chromatographic (HPLC) assay utilizing ultraviolet (UV) detection for the determination of bisnafide in human plasma was developed, validated, and applied to plasma samples from patients undergoing cancer therapy. Plasma samples, containing an internal standard, XE842, were first deproteinized with 2.0 ml acetonitrile, and subsequently, 1.0 ml of pH 9 boric acid–potassium chloride–sodium hydroxide buffer (0.1 M) was added. To this mixture, 9.0 ml of ethyl ether was added then vortex mixed. Following centrifugation, the ether layer was back-extracted into 250 μl of 0.1 M phosphoric acid, then removed by vacuum aspiration. A portion of the remaining acid layer was directly injected onto the HPLC. Bisnafide was quantified using a Shiseido Capcell Pak C8 HPLC column and ultraviolet detection (274 nm). The lower limit of quantification was 10 ng ml⁻¹ using 1.0 ml plasma. The intraday precision (RSD) ranged from 2.7 to 8.6% over a concentration range of 10–1000 ng ml⁻¹. The interday precision (RSD) ranged from 5.6 to 11.5%. Overall mean accuracy was ±5.2%. The drug was stable in frozen heparinized human plasma stored at −20°C for at least 1 year and stable throughout at least two freeze–thaw cycles. This method was successfully utilized for quantifying plasma concentrations needed to study the clinical pharmacokinetics of bisnafide in patients undergoing cancer therapy.     

Determination of endothelin antagonist BMS182874 in plasma by high-performance liquid chromatography

A simple and rapid high performance liquid chromatography (HPLC) method was developed for the determination of BMS182874 (BMS) in mouse plasma. The drug was extracted from plasma by a liquid-liquid extraction process. The method consists of reversed-phase chromatography using a Thermo Hypersil-Keystone RP-18 5 microm, 250 x 2.1 mm column and UV spectrophotometer detection at 255 nm. The mobile phase consists of 45% (v/v) acetonitrile: 55% (v/v) trifluoroacetic acid (0.015% v/v; pH 3.0) at a flow rate of 0.6 ml/min. Validity of the method was studied and the method was precise and accurate with a linearity range from 100 ng/ml to 1000 ng/ml. The extraction efficiency was found to be 81, 84 and 87% for 100, 500 and 1000 ng/ml, respectively for spiked drug in plasma. The limit of quantification and limit of detection were found to be 50 and 10 ng/ml, respectively in plasma. Within-day and between-day precision expressed by relative standard deviation was less than 4% and inaccuracy did not exceed 4%. The assay was also used to analyze samples collected during animal studies. The suitability and robustness of the method for in vivo samples were confirmed by analysis of BMS from mouse plasma and tissues dosed with BMS.     

Determination of ketorolac in human serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of ketorolac in human serum using a new extraction method with a good recovery. Human serum samples (1.0 ml) spiked with known concentrations of ketorolac tromethamine and 10 μg of ketoprofen as the internal standard (IS) were acidified with 200 μl of 1N HCl and extracted with 7 ml ofn-hexane-ether (7∶3 v/v). Extracts were centrifuged and organic layer was back-extracted with 400 μl of 0.1% tromethamine solution. Twenty μl of centrifuged aqueous layer was injected onto a reversed-phase octyl column and eluted with a mixture of acetonitrile, water, methanol, and triethylamine [35∶55∶10∶0.1 (v/v), pH 3.0] at a flow rate of 1.0 ml/min. Ultraviolet detection of ketorolac and IS was carried out at 300 nm. The calibration curve obtained using peak area ratios showed a good linearity (in concentration range 10–150 ng/ml r²=0.9944; in range 50–2000 ng/ml, r²=0.9998). The mean intra-day accuracy and precision for this HPLC method were found to be 3.6 and 3.7%, respectively. The mean inter-day accuracy and precision were found to be 4.0 and 3.7%, respectively, in the concentration range 50–2000 ng/ml. The recovery of ketorolac from serum was 92.0 (±5.7)% at the concentration of 100 ng/ml. This method proved to be readily applicable to the assay of ketorolac in human serum.     

Sensitivity improvement on detection of Coptidis alkaloids by sweeping in capillary electrophoresis

A simple and rapid sweeping method for the online improvement of detection sensitivity of the main alkaloids of Coptidis Rhizoma has been developed in this work. Optimum separation conditions were found as follows: electrophoretic running solution comprising 100 mM phosphoric acid, 15 mM sodium dodecyl sulfate (SDS) and 10% (v/v) tetrahydrofurane with pH 1.82; running voltage of -25 kV; sample matrix composed of 50 mM phosphoric acid and sample injection at 1000 mbar for 60 s (sample injection volume ca. 2.75 microl). With this sweeping method, the concentration limits of detection of berberine, coptisine and palmatine were found to be 2.5 ppb (ng/ml), which was about 500 times lower than those from conventional sample injections. Baseline separation was achieved for the main alkaloids within 15 min. After validation, the developed method was applied to determine the quantity of berberine, coptisine and palmatine in a Coptidis Rhizoma sample. The method should be able to be used in identification and quantitative evaluation of the crude drugs requiring only a minor amount of sample.     

Assay for nipecotic acid in small blood samples by gas chromatography-mass spectroscopy

An analytical method for nipecotic acid quantification in rat blood was developed utilizing a stable isotope internal standard and capillary gas chromatography–mass spectroscopy. The method involves a solid phase extraction step followed by a two-step derivatization. The analytes are separated by capillary gas chromatography and detected by selected ion monitoring of their base peaks at 180 and 185 m/z, respectively. The assay has a limit of detection (LOD) of 10 ng/ml and a limit of quantification of 26 ng/ml in 200 μl of rat whole blood. The linear range of the assay covers from 26 to 6500 ng/ml (r²=0.9996, n=9). The coefficient of variation was less than 10% at concentrations of 50, 1000 and 5000 ng/ml. The assay was used to characterize the pharmacokinetics of R-(-)-nipecotic acid in a rat. R-(-)-nipecotic acid clearance was 4.2 ml/min, its half-life was 1.5 h and its volume of distribution at steady state was 325 ml.