Solid-supported reagents for the simultaneous extraction and derivatization of carboxylic acids from aqueous matrices. Studies on optimization of reaction conditions

Solid-supported reagents for affecting a simultaneous extraction and derivatization of lipophilic and hydrophilic carboxylic acids from aqueous matrices were further investigated. The reagent consisted of pentafluorobenzyl bromide impregnated on the macroreticular resin XAD-2. New impregnation methods were developed which reduced the amount of pentafluorobenzyl bromide required and which were compatible with existing methods that utilize XAD-2 as an adsorbant. Factors affecting pentafluorobenzylation of these analytes were studied and the reaction conditions were optimized. Compatibility with simple biological samples was demonstrated using arachidonic acid as a model. Evidence was obtained suggesting that metal ions on the resin may be catalytic factors.     

Analysis of urinary biomarkers for exposure to alkyl benzenes by isotope dilution gas chromatography-mass spectrometry

A validated GC-MS method for the analysis of urinary metabolites of alkyl benzenes is reported. Metabolites for exposure to toluene, xylene and ethylbenzene were analyzed simultaneously using stable isotope substituted internal standards. The method entailed acidic deconjugation of urine samples followed by extractive alkylation with pentafluorobenzyl bromide as alkylating agent. The resulting pentafluorobenzyl derivatives of ortho-, meta-, para-cresol, mandelic acid (MA), hippuric acid (HA) and ortho-, meta-, para-methylhippuric acid (MHA) were then quantified by SIM. Optimized reaction conditions for the extractive alkylation step are reported. The derivatives were found to be sufficiently stable for overnight batch analysis. The LODs were below 0.1 micromol/L for the cresols and below 1 micromol/L for MA and the HAs. Within-batch precision for o-MHA was 7%, for m-MHA 5%, for p-MHA 5.2% and below 5% for the rest of the analytes.     

Determination of nalidixic acid by fluorometry with sodium borohydride and hydrogen peroxide

A simple and sensitive fluorometric method for the determination of nalidixic acid was established by using 0.75M sodium borohydride and 7.5% hydrogen peroxide solution as fluorogenic reagents. Analyte concentrations of 0.0232-11.6 pg/mL could be determined with high precision and accuracy by the method. A relative standard deviation of 1.75% was obtained for a nalidixic acid concentration of 0.232 pg/mL. The method was satisfactorily applied to the determination of nalidixic acid in human serum, fish muscle, and chicken muscle, and the calibration curves were linear from 0.23 to 58.00 pg/mL, from 9.28 to 32.48 mg/kg, and from 4.64 to 23.20 mg/kg, respectively. The specificity of the reaction is also discussed.     

Comparison of different methods for the determination of several quinolonic and cinolonic antibiotics in trout muscle tissue by HPLC with fluorescence detection

Summary Quinolonic and cinolonic derivatives are mainly used as antibacterials in fish-farms. In this paper we describe a careful revision of the treatment procedures of samples and a prodedure for the determination of residues of these compounds. Because of the complexity and duration of these procedures, several studies have been carried out and these have lead to a simpler and shorter method. Three approaches have been examined: lyophilization followed by extraction with chloroform, solid-liquid extraction with chloroform and solid-liquid extraction with sodium hydroxide solution, followed by liquid-liquid partition in chloroform. Some previous studies into the partition equilibrium are also included. As a result of our studies we propose a procedure with a lower number of steps than those previously described in the literature. This method has been applied to the analysis of nalidixic, 7-hydroxymethylnalidixic and oxolinic acids and cinoxacin in trout muscle. These analysis have been carried out using an HPLC system equipped with a C₁₈ column and fluorimetric detection. The mobile phase was acetonitrile:oxalic acid. The recoveries obtained were: 70–97% for 7-hydroxymethylnalidixic acid, 75–78% for nalidixic acid, 71–95% for oxolinic acid and 72–85% for cinoxacin.     

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 11 (fluoro)quinolone antibiotics in swine kidney

A LC-MS-MS method has been validated for the simultaneous quantification of 11 (fluoro)quinolone antibiotics at the maximum residue level (MRL) in swine kidney. The studied compounds were danofloxacine, cinoxacine, ciprofloxacine, noxacine, enrofloxacine, flumequine, marbofloxacine, nalidixic acid, norfloxacine, ofloxacine and oxolinic acid. The method involves solid-phase extraction of these compounds followed by LC-MS-MS analysis using an electrospray ionisation interface. Limits of quantification < or = 50 microg/kg could be obtained in swine kidney, much lower than every MRL. The validation is discussed. This work was carried out in order to support the European Union policy on consumer health     

Gradient HPLC of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and pharmaceutical samples using C-18 and RP-amide columns

A simple and highly sensitive high pressure liquid chromatographic (HPLC-UV) method has been developed for the determination of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, in mobile phase citrate buffer (0.001 M) of pH 4.5 prepared in water (X), methanol (Y), and ACN (Z) using gradient at a flow rate of 1.0 mL/min by direct UV absorbance detection at lambda = 280 nm. Separation of analytes was studied on the C-18 and RP-amide columns and best results were observed on the RP-amide column with LODs (3.3 x S/m) 0.89, 0.55, 0.67, and 1.41 ng/mL for ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, respectively, and better RSD than the C-18 column. The recovery of Fluoroquinolones (FQs) in urine, ground water, hospital wastewater, and chicken muscle using this method is more than 90%. The method was successfully applied to the analysis of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid in urine, ground water, pharmaceutical dosage forms, hospital wastewater, and chicken muscle.     

Influence of tryptophan loading on urinary excretion of anthranilic acid and 3-hydroxyanthranilic acid by men and women as determined by alkali flame ionization gas chromatography

A gas chromatographic method with alkali flame ionization detection is described for the determination of urinary total (free and conjugated) anthranilic acid (AA) and 3-hydroxyanthranilic acid (HAA) as their pentafluorobenzyl esters. Prior to analysis, urine was hydrolysed using hydrochloric acid in a boiling water bath. The highest AA and HAA yields were obtained with 4 M hydrochloric acid and a hydrolysis time of 4 h. The coefficients of variation of the between-run analyses of AA and HAA at the endogenous level were 7.2 and 5.8%, respectively. The average recovery for both substances was 84%. The method described has been used to study the excretion of AA and HAA in the urine of healthy males and females before and after an oral load of tryptophan. Furthermore, the influence of oral contraceptives has been investigated. Results indicate that for both sexes the excretion of AA in the urine was higher than that of HAA, except after tryptophan loading. The excretion of AA by women was higher than by men. For HAA, the results of both sexes were comparable. Furthermore, for neither of the sexes was a diurnal variation of AA or HAA observed. After tryptophan loading, the formation of HAA was increased by more than that of AA. Results obtained for women on oral contraceptives indicate a hormonal-induced inhibition of AA formation.     

Direct determination of nalidixic acid in urine by matrix isopotential synchronous fluorescence spectrometry

A new method for the determination of nalidixic acid in urine is proposed for concentrations between 25 and 1000 ng ml(-1) by means of matrix isopotential synchronous fluorescence spectrometry. This new technique is useful for the determination of compounds in samples with unknown background fluorescence, such as nalidixic acid in urine, without the need for tedious preseparation. The method was performed in ethanol/water medium (80% v/v), at an apparent pH of 2.9 provided by adding sodium monochloracetate/monochloroacetic acid buffer solution. The method was successfully applied to the determination of nalidixic acid in urine. Better sensitivity and reproducibility are achieved in these matrices than with the fluorimetric methods described in the literature.     

Simultaneous Determination of 13 Phenoxy Acid Herbicide Residues in Soybean by GC‐ECD

Abstract

In this study, a gas chromatography method for the multiresidue determination of 13 phenoxy acid herbicide residues (4‐CPA, 4‐CPP, phenoxy butyric acid, dicamba, MCPA, MCPP, MCPB, 2,4‐D, 3,4‐D, 2,4,5‐T, 2,4,5‐TP, 2,4‐DP, and 2,4‐DB) in soybean was developed. After the removal of fat using n‐hexane, the sample was extracted with acetonitrile‐50 mM HCl (v/v 7:3), and then followed by liquid‐liquid partition with n‐hexane (saturated by acetonitrile). The soybean extract was further cleaned up by anion exchange column. Then the residues were derived with pentafluorobenzyl bromide and the resulting pentafluorobenzyl (PFB) esters were analyzed by a gas chromatograph equipped with an electron capture detector (ECD), quantified by the external standard method. Recoveries of spiked samples at two fortified levels (0.01 and 0.1 mg/kg) are all above 70%. And the relative standard deviation was less than 20%. It shows that the limits of determination (LOD) of this method (S/N≥3) can meet the requirement of maximum residue analysis in import/export inspection for soybean.     

Studies on steroids. CCXXVIII Trace analysis of bile acids by gas chromatography-mass spectrometry with negative ion chemical ionization detection

A suitable derivatization method for the trace analysis of bile acids by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Of various derivatives for the carboxyl group, the pentafluorobenzyl (PFB) ester provided the highest value of the ratio of the negative to the positive ion current. A characteristic carboxylate anion [M - 181]- was produced as the most abundant ion by the loss of the PFB group in NICI. The PFB esters were further derivatized to the dimethylethylsilyl (DMES) ethers, whereby lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid and cholic acid were distinctly separated by GC on a cross-linked methyl silicone fused-silica capillary column. The detection limit for the PFB-DMES derivatives of dihydroxylated bile acids was 2 fg when the fragment ion was monitored at m/z 563 in the NICI mode using isobutane as a reagent gas.     

Determination of polysulphides in blood by gas chromatography and gas chromatography-mass spectrometry.

A sensitive and simple method to determine polysulphides in human blood, using an extractive alkylation technique and gas chromatography, has been devised. Polysulphides were alkylated with pentafluorobenzyl bromide, and then converted into bis(pentafluorobenzyl)disulphide by desulphuration with potassium cyanide. The disulphide was analysed qualitatively by mass fragmentography and quantitatively by gas chromatography with electron-capture detection. The lower limit of detection was 0.005 mumol/ml. Field testing in a suicide case confirmed the validity of the method.     

Determination of lovastatin acid in serum by gas chromatography/mass spectrometry

The acid form of lovastatin, an HMG-CoA reductase inhibitor, was analyzed by gas chromatography/negative-ion chemical ionization mass spectrometry after derivatization with pentafluorobenzyl bromide and bis-(trimethylsilyl)trifluoroacetamide (BSTFA). Mass spectrometry of this derivative produced a dominant [M-181]- ion under chemical ionization conditions using ammonia as the reagent gas. The limit of detection was approximately 2 pg injected on column.     

Capillary gas chromatography/negative ion chemical ionization mass spectrometry for the quantification of bile acids including 1 beta-hydroxylated and unsaturated bile acids in serum and urine

12 bile acids, including 1 beta-hydroxylated and unsaturated bile acids, have been quantified by capillary gas chromatography/negative ion chemical ionization mass spectrometry, using the trimethylsilyl(TMS) ether derivatives of bile acid pentafluorobenzyl(PFB) esters. The analysis time is 12 min and the minimum measurable amount is 100 fg for each bile acid. Bile acids in 200 microL of serum and 50 microL of urine from healthy human adults were measured. These small sample sizes enhance the practicality of using this method as a screening test for bile acids in the serum and urine of human infants, where small sample size is a major problem.     

Micro-quantitative determination of ciprostene in plasma by gas chromatography-mass spectrometry coupled with an antibody extraction.

A simple and highly sensitive method has been developed for the determination in plasma of ciprostene, 9 beta-methyl-6 alpha-carbaprostaglandin I2, using gas chromatography-mass spectrometry following solid-phase extraction on an immobilized antibody column. The anti-ciprostene antibody obtained from rabbit serum was coupled to an agarose support matrix, and the immobilized antibody thus prepared was used as an extraction phase for sample clean-up. The extracted drug was treated with pentafluorobenzyl bromide followed by bis(trimethylsilyl)trifluoroacetamide. The derivative was quantitatively analysed by negative-ion chemical ionization gas chromatography-mass spectrometry. The lower limit of quantitation was 50 pg/ml when 1 ml of human plasma was used. The plasma concentration of ciprostene in a dog treated with ciprostene at 2.5 micrograms/kg was determined successfully by this method.     

Determination of resin and fatty acids in sediments near pulp mill locations

A gas chromatographic method for the determination of resin and fatty acids in sediments is described. In this procedure, the sediment sample was air-dried and soxhlet-extracted with a mixture of acetone--methanol (88:12, v/v) in the presence of hydrochloric acid. The acids extracted were converted into their pentafluorobenzyl esters and were then cleaned up on a deactivated silica gel column. Final analysis was performed on either a DB-17 or a DB-5 capillary column with electron-capture detection. Quantitative recovery was obtained from fortified sediments for all acids except palustric, neoabietic and levopimaric acids. The detection limit of all acids in this method was 0.1 micrograms/g based on 1 g of sample. This procedure has been successfully applied to the monitoring of resin and fatty acids in sediment samples collected in the vicinity of several Canadian pulp mill locations.     

Determination of pentafluorobenzyl derivatives of phosphonic and phosphonothioic acids by gas chromatography-mass spectrometry

A method based on gas chromatography-mass spectrometry (GC/MS) has been evaluated and standardized for the analysis of pentafluorobenzyl (PFB) derivatives of alkylphosphonic, O-alkyl alkylphosphonic and phosphonothioic acids. The pentafluorobenzyl (PFB) derivatives are much more stable as compared to the conventionally used trimethylsilyl derivatives. The conditions for the derivatization and analysis have been optimized to achieve the best detection limits in negative chemical ionization (NCI) mode.     

Analysis of pipecolic acid in biological fluids using capillary gas chromatography with electron-capture detection and [2H11]pipecolic acid as internal standard.

A sensitive and accurate stable isotope dilution assay was developed for the measurement of pipecolic acid in body fluids using capillary gas chromatography with electron-capture detection. The method utilizes [2H11]pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous solution (pH 11.5) of the amine moiety with methyl chloroformate to the N-methylcarbamate, followed by acidic ethyl acetate extraction at pH less than or equal to 2 and further derivatization of the carboxyl moiety with pentafluorobenzyl bromide, the excess of which was removed by solid-phase extraction. Control values have been determined in the plasma of at-term infants, age greater than 1 week (n = 21, mean = 1.36 microM, range = 0.47-3.27 microM). The utility of the method was demonstrated by quantitating pipecolic acid in biological fluids derived from patients with peroxisomal disorders. The method was validated against an established electron-capture negative ion mass fragmentographic technique.     

尿中3种苯氧羧酸类除草剂的气相色谱分析法

本文研究了2,4-滴、2,4-滴丙酸和2甲4氯3种苯氧羧酸类除草剂用硫酸、三氯化硼、氯化氢和三氟乙酸等4种催化剂的甲醇、乙醇、丙醇、丁醇、戊醇、苯甲醇、三氟乙醇、五氟丙醇、二氯丙醇和五氟苯甲醇等10种醇的酯化衍生反应条件,在此基础上建立了尿中3种苯氧羧酸类除草剂的各种衍生化气相色谱电子俘获检测方法,其中较灵敏的方法2,4-滴和2,4-滴丙酸的检出限低于10 ng/mL,2甲4氯的检出限低于20ng/mL,适于职业接触者和中毒者的尿分析。     

Extraction and quantitative analysis of 1-aminocyclopropane-1-carboxylic acid in plant tissue by gas chromatography coupled to mass spectrometry

We developed a new method for the determination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) using quantitative GC-negative chemical ionisation MS as a detection and quantification system, in combination with isotope dilution using [2H4]ACC and an off-line solid-phase extraction. By derivatisation with pentafluorobenzyl bromide, ACC could easily be detected with m/z 280 being the most abundant ion. Determination of this component resulted in a detection limit of 10 fmol and a linear fit in the 100 fmol-100 pmol range. The combination of a rapid, high yield purification method with a stable derivatisation procedure and a sensitive detection method allowed the detection of ACC in samples as low as 100 mg fresh mass.     

液相色谱-串联质谱法检测水产品中15种喹诺酮类药物残留量

建立了液相色谱-串联质谱技术同时检测水产品中15种喹诺酮类药物(氟罗沙星、氧氟沙星、依诺沙星、诺氟沙星、环丙沙星、恩诺沙星、洛关沙星、单诺沙星、奥比沙星、双氟沙星、沙拉沙星、司帕沙星、口恶喹酸、萘啶酸、氟甲喹)残留量的方法.试样中残留的喹诺酮类药物采用乙腈提取,提取液经正已烷液液分配脱脂后,以强阳离子固相萃取小柱净化,液相色谱.串联质谱法测定.对液/质分离条件与样品前处理条件进行了优化,并对喹诺酮类药物在分析过程的稳定性进行了研究.15种喹诺酮类药物在1.0～100 μg/L范围内线性关系良好,相关系数为0.9924～0.9992.在0.002～0.04 mg/kg浓度范围内,平均加标回收率在79.9%～93.8%;相对标准偏差为4.8%～14.6%.方法可满足水产品中喹诺酮类药物多残留检测与确证的需要.