High-performance liquid chromatographic determination of pyrazinamide in cerebrospinal fluid and plasma in the rabbit

A simple procedure for the determination of pyrazinamide in plasma and cerebrospinal fluid in the rabbit is described. The assay involves a preliminary extraction of the drug and an internal standard, paracetamol, from the acidified sample (pH 4.2). The extract is evaporated to dryness at 45 degrees C and the residue is redissolved in methanol (50 microliters). A 25-microliters aliquot is injected into the liquid chromatograph and eluted with acetonitrile-10 mM phosphate buffer of pH 3.5 (10:90, v/v) on a 30-microns C8 pre-column linked to a 5-microns C8 reversed-phase column at ambient temperature (25 +/- 1 degree C). The eluate is detected at 215 nm. The method has been used to investigate the disposition of pyrazinamide in plasma and cerebrospinal fluid in six rabbits.     

Simultaneous determination of some active ingredients in cough and cold preparations by gas chromatography, and method validation

A simple and rapid gas chromatographic (GC) method has been developed for the simultaneous determination of combinations of acetaminophen, phenylpropanolamine hydrochloride, guaifenesin, pseudoephedrine hydrochloride, caffeine, chlorpheniramine maleate, and dextromethorphan hydrobromide in cough and cold tablets and syrups. After extraction of the analyte with alkaline ethyl acetate, 2 microL extract was injected (splitting ratio of 50:1) into a gas chromatograph equipped with a CBP1-M25-025 fused silica capillary column (25 m x 0.22 mm; film thickness, 0.25 microm). The column temperature was held at 150 degrees C for 5 min, increased to 175 degrees C at 3 degrees C/min, and increased to 270 degreesC at 10 degrees C/min. The temperatures of the flame ionization detector and injector were maintained at 300 degrees C. The GC method is inexpensive, rapid, accurate, and precise, and thus it can be used for routine analysis of tablet and syrup preparations in quality control laboratories of pharmaceutical companies.     

The use of formic acid in carrier gas. Rapid method for identification and determination of phytanic acid by gas chromatography-mass spectrometry-chemical ionization

A rapid gas chromatographic method to determine phytanic acid in plasma from Refsum's disease is described. After a brief alkaline hydrolysis of lipids, the biological sample is directly injected into a glass pre-column; an acid carrier gas (formic acid in nitrogen) is used to displace the long-chain fatty acids from their sodium salts and from their binding to proteins. Formic acid introduced through the column may also be used as a reagent gas for chemical ionization in combined gas chromatography--mass spectrometry; fatty acids (C14 to C18:2 and phytanic acid) are easily identified by their M + 1 (base peak) and M - 17 peaks. The described procedure is also suitable for studying normal fatty acids from plasma lipids.     

Fluorimetric flow-injection analysis of total amounts of aldehydes in auto exhaust gas and thermal degradation emission gas with cyclohexane-1,3-dione

A simple flow-injection (FI) spectrofluorimetric method for the assay of total volatile aldehydes in auto exhaust gas and emission gas from thermal degradation was developed. Aldehydes, such as formaldehyde, acetaldehyde, propionaldehyde and n-butyraldehyde, reacted with cyclohexane-1,3-dione (CHD) to form more strongly fluorescent compounds. A two-channel flow system was assembled. Distilled water and 0.02% CHD were delivered at 0.75 ml min(-1). The optimum conditions were pH 5 (2.2 M CH(3)COONH(4)-CH(3)COOH buffer solution), reaction temperature 70 degrees C, reaction coil length 0.5 mm i.d. x 7 m, cooling coil length 2 m, sample size 60 microl, excitation and emission wavelengths, 376 nm and 452 nm. Aldehydes in sample gas (10 1) were collected by passing the gas at a flow rate of 0.5 1 min(-1) through two impingers connected in series. 10 ml of methanol was used as an absorbent and diluted sample solution was injected into the carrier stream. The calibration graph was linear in the range 100-1000 ppb. The detection limit was 30 ppb and a sampling frequency of 30 h(-1) was attained. Relative standard deviation for 10 standard formaldehyde solutions (500 ppb) was 1.5%. This rapid and simple FI method was applied to the determination of the total amount of aldehydes, calculated as formaldehyde, in auto exhaust gas and emission gas from the thermal degradation of polymers. The method is useful for monitoring aldehyde emissions and investigating the removal effect of aldehydes from various sources.     

Simultaneous determination of pseudoephedrine hydrochloride and diphenhydramine hydrochloride in cough syrup by gas chromatography (GC)

A simple, rapid and precise gas chromatographic method has been developed for the simultaneous determination of pseudoephedrine hydrochloride and diphenhydramine hydrochloride in cough syrup, using a SS column of 10% OV 1 on chromosorb W-HP (80-100 mesh) and nitrogen as a carrier gas at a flow rate of 30 ml min(-1). The oven temperature was programmed at 135 degrees C for 1 min, with a rise of 10 degrees C min(-1) up to 250 degrees C (held for 5 min). The injector and detector port temperatures were maintained at 280 degrees C. Detection was carried out using Flame ionization detector. Guaphenesin was used as an internal standard. Results of assay and recovery studies were statistically evaluated for its accuracy and precision.     

Determination of atropine in biological specimens by high-performance liquid chromatography

A sensitive and selective method for the determination of atropine in biological specimens has been developed. Samples alkalinized with sodium hydroxide were extracted with dichloromethane, and the organic phase was evaporated in a water-bath at 50 degrees C for ca. 10 min. The residue was dissolved in the mobile phase and injected into a reversed-phase column (TSK gel ODS-120A). The retention time for atropine could be varied by changing either the acetonitrile-water ratio in the mobile phase or the pH of the mobile phase. Acetonitrile-water (2:8, v/v) containing 6 mM phosphoric acid was used as mobile phase. Samples of 200 microliters or less were injected into the chromatography and measured at 215 nm. The recoveries of atropine added to drug-free specimens were satisfactory with coefficients of variation of 4% or less. Ninety-two compounds tested did not interfere with the assay of atropine. The method has been applied for monitoring atropine concentrations in cases of organophosphate and drug poisoning.     

Simultaneous determination of carbamazepine and its epoxide metabolite in plasma and urine by high-performance liquid chromatography

A simple procedure for the simultaneous determination of carbamazepine and its major metabolite, carbamazepine epoxide, in plasma and urine is described. The assay involves two extractions of the drugs and an internal marker, clonazepam, from the alkalinized sample. The extract is evaporated to dryness at 45 degrees C and the residue is redissolved in methanol (30 microliters). A 25-microliters aliquot is injected into the liquid chromatograph and eluted with acetonitrile-water (40:60, v/v) on a C18 pre-column linked to a 5-microns C8 reversed-phase column. The eluent is detected at 215 nm. The method has been used to investigate the steady-state concentrations of carbamazepine and carbamazepine epoxide in the plasma and urine of a manic-depressive patient.     

Development of a low volume plasma sample precipitation procedure for liquid chromatography/tandem mass spectrometry assays used for drug discovery applications

The demand for high sensitivity bioanalytical methods has dramatically increased in the drug discovery stage; in addition, there has been a growing trend of reducing the sample volume that is required for these assays. A sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedure has been developed and tested to meet these needs. The assay requires only a low plasma sample volume (10 microL) and employs a protein precipitation procedure using a 1:6 plasma/acetonitrile ratio. The supernatant is injected directly into the LC/MS/MS system using the selected reaction monitoring (SRM) procedure for detection. A generic HPLC gradient based on a methanol/water mobile phase with a flow rate set to 0.8 mL/min was used. The test method showed very good linearity between 0.1-1000 ng/mL (R2 = 0.9737), precision (%RSD = 6-9), accuracy (%RE = -2) and reproducibility (%RSD = 11). A drug discovery IV/PO study was assayed using both the new low volume method and our standard volume (50 microL) method. The correlation of the two sets of data from the two methods was excellent (R2 = 0.9287). This new assay procedure has been successfully used in our laboratory for over 100 different rat or mouse discovery PK studies.     

Luminol-dependent chemiluminescence analysis of human platelets

We describe a simple, sensitive, and reproducible assay system to measure the chemiluminescence (CL) produced by injecting arachidonic acid (AA) into a preparation of human platelets containing luminol.The CL appears to result from the metabolism of the AA by enzymes in human platelets, namely, cyclooxygenase, lipoxygenase, and possibly peroxidases. It is believed that when the AA is injected, free radicals and/or oxidizing agents are formed that react with the luminol producing an excited state and emitting blue light (425 nm).The enzymes can be inhibited by drugs to varying degrees. BW755C inhibits all CL at micromolar doses and it is known to inhibit both lipoxygenase and cyclooxygenase. Aspirin, indomethacin, and sulindac sulfide inhibit only cyclooxygenase and inhibit 35–65% of the light from an individual. This assay system can be used to screen certain drugs that are effective in inflammatory diseases. It could be used to determine whether the drugs would be effective in a given individual and also whether drugs have a long-term toxic effect in vivo on platelets. Further the assay is practical with a few milliters of blood.     

A new method for the determination of residual piperazine in pharmaceuticals by capillary gas chromatography

A new selective open tubular capillary gas chromatographic method is developed for the determination of trace amounts of piperazine. Piperazine is extracted from pharmaceuticals into cyclohexane and is partitioned with water. The aqueous solution is then injected into a 5% crosslinked Ph-Me silicone column programmed at 50-180 degrees C for 10 min. Piperazine is eluted after 3.18 min under isothermal conditions. The lower limit of determination is 0.4 ppm. This method has been successfully applied for the assay of piperazine in pharmaceutical formulations and its trace determination in fluoroquinolone drugs such as norfloxacin and ciprofloxacin. The method is reproducible and the standard and relative standard deviation for 10 repeated injections of 2 mug piperazine are 0.7 and +/-2.1% respectively.     

Preconcentration and determination of cadmium by GFAAS after solid-phase extraction with synthetic zeolite.

The solid-phase extraction (SPE) method for the preconcentration of trace amounts of cadmium using synthetic zeolite A-4 and its determination by graphite furnace atomic absorption spectrometry (GFAAS) was investigated. The preconcentration conditions, such as the optimum pH range of the sample solution for the adsorption of cadmium and the kind of acid solution for dissolving the cadmium-adsorbed synthetic zeolite A-4, as well as the measurement conditions for the determination of cadmium by GFAAS, e.g., the ashing and atomizing temperature, were investigated. Quantitative recovery of cadmium onto zeolite A-4 from the sample solution over the pH range 2.0 - 9.0 was achieved by the batch method. After the solid-phase (cadmium-adsorbed zeolite A-4) was separated from the sample solution by a membrane filter, it was dissolved in 2.0 cm(3) of 2.0 mol dm(-3) nitric acid. An aliquot of the resulting solution was injected into the graphite furnace. In GFAAS measurements an alternate gas (Ar, 90%; O(2), 10%) was used as a sheath gas, and the ashing temperature and atomizing temperature were 400 degrees C and 1600 degrees C, respectively. The detection limit (3 sigma) for cadmium was 0.002 microg dm(-3). The relative standard deviation at 0.010 microg dm(-3) was 3.5 - 4.5% (n = 5). The proposed method has been successfully applied to the analysis of trace cadmium in environmental water samples.     

气相色谱法测定新型多元复合液肥中二羧酸

 建立了新型多元复合液肥中丁二酸、戊二酸、己二酸的气相色谱分析方法。色谱条件：５％ＳＥ－３０不锈钢填充柱,程序升温,内标物为丙二酸二乙酯,３种二元酸二乙酯分别在２．３～９．３μｇ,１０．８～４３．２μｇ和２．８～１１．０μｇ质量范围内,其样品质量与样品峰面积和内标峰面积之比呈良好的线性关系,相关系数ｒ≥０．９９５５。     

Liquid chromatographic assay for the cyclic depsipeptide aplidine, a new marine antitumor drug, in whole blood using derivatization with trans-4'-hydrazino-2-stilbazole

A sensitive bio-analytical assay for the depsipeptide aplidine in plasma has been modified and tested for human whole blood samples. The adapted method is based on reversed-phase liquid chromatography and fluorescence detection of the trans-4'-hydrazino-2-stilbazole derivative of the analyte. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, the derivatization with the hydrazino reagent is performed in a water-acetonitrile mixture at pH = 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2-100 ng/mL range, with 2 ng/mL being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The stability of aplidine in whole blood at ambient temperature and at 37 degrees C is limited; recoveries in the range 60-85% were observed after 7 h. Further, adequate stability of aplidine in plasma at -80 and -20 degrees C for 35 months could now be demonstrated.     

Dispersive liquid-liquid microextraction method based on solidification of floating organic drop combined with gas chromatography with electron-capture or mass spectrometry detection

A simple dispersive liquid-liquid microextraction (DLLME) method based on solidification of a floating organic drop (DLLME-SFO) technique combined with gas chromatography/electron-capture detection (GC/ECD) or gas chromatography/mass spectrometry (GC/MS) has been developed. The proposed method is simple, low in cost, and of high precision. It overcomes the most important problem in DLLME, the high-toxic solvent used. Halogenated organic compounds (HOCs) in water samples were determined as the model compounds. The parameters optimized for the DLLME-SFO technique were as follows: A mixture of 0.5 mL acetone, containing 10 microL 2-dodecanol (2-DD-OH), was rapidly injected by syringe into the 5 mL water sample. After centrifugation, the fine 2-DD-OH droplets (8+/-0.5 microL) were floated at the top of the screwcap test tube. The test tube was then cooled in an ice bath. After 5 min the 2-DD-OH solvent had solidified and was then transferred into a conical vial; it melted quickly at room temperature and 3 microL (for GC/ECD) or 2 microL (for GC/MS) of it was injected into a gas chromatograph for analysis. The limit of detection (LOD) for this technique was 0.005-0.05microgL(-1) for GC/ECD and was 0.005-0.047 microgL(-1) for GC/MS, respectively. The linear range of the calibration curve of DLLME-SFO was from 0.01 to 500 microgL(-1) with a coefficient of estimation (r2)>0.996 for GC/ECD and was from 0.02 to 500 microgL(-1) with a coefficient of estimation (r2)>0.996 for GC/MS.     

Headspace gas chromatographic determination of beta-galactosidase activity using electron-capture detection

A headspace gas chromatographic method for the determination of beta-galactosidase (E.C. 3.2.1.23) activity is described. The method, in which 2,2,2-trichloroethyl beta-D-galactopyranoside (beta-TCG) is used as substrate, involves determination of the liberated 2,2,2-trichloroethanol by gas chromatography with electron capture detection. The preparation of beta-TCG and of 2,2,2-trichloroethyl alpha-D-galactopyranoside is described. A Km = 0.80 mM was found for the enzymatic hydrolysis of beta-TCG employing beta-galactosidase from Escherichia coli. The assay has been applied to the quantitative determination of E. coli bacteria.     

Semicarbazide is a minor thermal decomposition product of azodicarbonamide used in the gaskets of certain food jars

Evidence is presented for the first time showing that semicarbazide (SEM) is a minor thermal decomposition product of the blowing agent azodicarbonamide (ADC). A novel direct analytical method based on liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) has been developed to determine SEM in foamed polyvinyl chloride (PVC) seals of metal lids, as well as in commercially available ADC. The direct LC-MS/MS method for gaskets entails extraction of the gaskets in hot water, addition of ((15)N(2)(13)C)-SEM as internal standard, and injection of an aliquot directly into the LC-MS system, achieving good sensitivity (S/N = 348 for 2 ng injected on-column) and monitoring three characteristic mass transitions (m/z 76-->31; 76 -->44; 76-->59). Semicarbazide can be detected in thermally treated ADC, reaching up to 0.93 mmol mol(-1) at 220 degrees C, as determined by the direct LC-MS/MS method. This new method is also compared to the classical derivatization method using 2-nitrobenzaldehyde (2-NBA) that is routinely employed to determine SEM as an indicator of the usage of the antimicrobial drug nitrofurazone, the use of which is not authorized in the European Union (EU). Both methods revealed proportional results, with approx. 3-fold higher levels recorded by the direct SEM approach, probably due to differences in the extraction procedures used. A limited survey of plastic seals from used press twist and twist-off metal lids on food jars (non-foamed and foamed) revealed levels of SEM ranging from 2 to 8689 microg kg(-1)(average = 1593 microg kg(-1), n= 57 determinations).     

Gas chromatographic analysis of urinary dimercaptosuccinic acid

The therapeutic use of disulfhydryl compounds such as 2,3-dimercaptosuccinic acid (DMSA) for the treatment of heavy metal poisoning has generated a requirement for specific and sensitive methods to determine those compounds in biological media. We have developed a gas chromatographic assay for DMSA in urine. The use of capillary column technology eliminates the requirement for a preliminary clean-up step. Samples are first reduced electrochemically to liberate DMSA present as disulfides. The reduced product is then extracted into ethyl acetate and the organic phase removed by evaporation. The residue is derivatized with N,O-bis (trimethylsilyl) acetamide for gas chromatography. The silylated DMSA derivative is then detected with a flame ionization detector. The detection limit for DMSA is 1.9 nmol per 1-microliter aliquot of derivatized extract injected on column (detector sensitivity at 1.10(-11) A/mV). The utility of the method was demonstrated by analyzing the urine of rats orally dosed with DMSA.     

Determination of acrylamide formed in asparagine/D-glucose maillard model systems by using gas chromatography with headspace solid-phase microextraction

A gas chromatographic method, along with a headspace solid-phase microextraction (HS-SPME), was developed for the determination of acrylamide formed in Maillard reaction model systems. The developed method was validated by liquid chromatography/mass spectrometry. A headspace sample was collected from an aqueous acrylamide solution (100 microg/mL) by SPME and directly injected into a gas chromatograph equipped with a nitrogen-phosphorus detector. The recovery of acrylamide from an aqueous solution was satisfactory, i.e, >93% under the conditions used. Acrylamide formed in an asparagine/D-glucose (molar ratio, 1/2) Maillard reaction model system heated at 150 and 170 degrees C for 20 min was collected and analyzed by the newly developed method using gas chromatography with nitrogen-phosphorus detection and HS-SPME. The amounts of acrylamide were 318 +/- 33 microg/g asparagine from a sample heated at 150 degrees C and 3329 +/- 176 microg/g asparagine from a sample heated at 170 degrees C. Addition of cysteamine or glutathione to the above model system reduced acrylamide formation. Acrylamide formation was not observed when cysteamine or glutathione was added to asparagine in the above model systems to obtain equimolar concentrations of both compounds. This newly developed method is simple and sensitive, and requires no solvent extraction.     

Determination of gas-oil miscibility conditions by interfacial tension measurements

Processes that inject gases such as carbon dioxide and natural gas have long been and still continue to be used for recovering crude oil from petroleum reservoirs. It is well known that the interfacial tension between the injected gas and the crude oil has a major influence on the efficiency of displacement of oil by gas. When the injected gas becomes miscible with the crude oil, which means that there is no interface between the injected and displaced phases or the interfacial tension between them is zero, the oil is displaced with maximum efficiency, resulting in high recoveries. This paper presents experimental measurements of interfacial tension between crude oil and natural gases (using a computerized drop shape analysis technique) as a function of pressure and gas composition at the temperature of the reservoir from which the crude oil was obtained. The point of zero interfacial tension was then identified from these measurements by extrapolation of data to determine minimum miscibility pressure (MMP) and minimum miscibility composition (MMC). The gas-oil miscibility conditions thus obtained from interfacial tension measurements have been compared with the more conventional techniques using slim-tube tests and rising-bubble apparatus as well as predictive correlations and visual observations. The miscibility pressures obtained from the new VIT technique were 3-5% higher than those from visual observations and agreed well with the slim-tube results as well as with the correlations at enrichment levels greater than 30 mol% C2+ in the injected gas stream. The rising bubble apparatus yielded significantly higher MMPs. This study demonstrates that the VIT technique is rapid, reproducible, and quantitative, in addition to providing visual evidence of gas-oil miscibility.     

Hollow fiber liquid phase microextraction combined with electrothermal atomic absorption spectrometry for the speciation of arsenic (III) and arsenic (V) in fresh waters and human hair extracts

A new method of hollow fiber liquid phase microextraction (HF-LPME) using ammonium pyrrolidine dithiocarbamate (APDC) as extractant combined with electrothermal atomic absorption spectrometry (ETAAS) using Pd as permanent modifier has been described for the speciation of As(III) and As(V). In a pH range of 3.0-4.0, the complex of As(III)-APDC complex can be extracted using toluene as the extraction solvent leaving As(V) in the aqueous layer. The post extraction organic phase was directly injected into ETAAS for the determination of As(III). To determine total arsenic in the samples, first As(V) was reduced to As(III) by l-cysteine, and then a microextraction method was performed prior to the determination of total arsenic. As(V) assay was based on subtracting As(III) form the total arsenic. All parameters, such as pH of solution, type of organic solvent, the amount of APDC, stirring rate and extraction time, affecting the separation of As(III) from As(V) and the extraction efficiency of As(III) were investigated, and the optimized extraction conditions were established. Under optimized conditions, a detection limit of 0.12 ng mL⁻¹ with enrichment factor of 78 was achieved. The relative standard deviation (R.S.D.) of the method for five replicate determinations of 5 ng mL⁻¹ As(III) was 8%. The developed method was applied to the speciation of As(III) and As(V) in fresh water and human hair extracts, and the recoveries for the spiked samples are 86-109%. In order to validate the developed method, three certified reference materials such as GBW07601 human hair, BW3209 and BW3210 environmental water were analyzed, and the results obtained were in good agreement with the certified values provided.