Automated derivatization and analysis of malondialdehyde using column switching sample preparation HPLC with fluorescence detection

Analyte derivatization is advantageous for the analysis of malondialdehyde (MDA) as a biomarker of oxidative stress in biological samples. Conventionally, however, derivatization is time consuming, error-prone and has limited options for automation. We have addressed these challenges for the solid phase analytical derivatization of MDA from small volume tissue homogenate samples. A manual derivatization method was first developed using Amberlite XAD-2 (12 mg) as the solid phase. Subsequently an automated column switching process was developed that provided simultaneous derivatization and extraction of the MDA-DH hydrazone product on a cartridge packed with XAD-2, followed by quantitative elution of the product to an analytical LC column (Waters NovoPak C18, 3.9 x 150 mm). The LOD was 0.02 microg/mL and recovery was quantitative. The method was linear (r(2) >0.999) with precision < 5% from the LOQ (0.06 microg/mL) to at least 35 microg/mL. The method was successfully applied to the analysis of small volume (30 microL) mouse tissue homogenate samples. Endogenous levels of MDA in the tissues ranged from 20 to 40 nmol/g tissue (ca. 0.1-0.2 microg/mL homogenate). Compared to conventional MDA analyses, the current method has advantages in automation, selectivity, precision and sensitivity for analysis from very small sample volumes.     

Determination of piperazine in working atmosphere and in human urine using derivatization and capillary gas chromatography with nitrogen- and mass-selective detection

A reliable routine method is presented for the determination of piperazine down to the sub-ppm level in aqueous solutions and in urine. The method includes a two-phase derivatization procedure with ethyl- or isobutyl chloroformate as the reagent, followed by a capillary gas chromatographic determination using nitrogen- or mass selective detection. The addition of ammonia ensured a quantitative recovery. Detection limits for piperazine in urine were ca. 20 ng/ml using nitrogen-selective and ca. 1 ng/ml with mass-selective detection. The calibration plots were linear in the investigated range, 100-10,000 ng/ml with nitrogen-selective and 30-3000 ng/ml with mass-selective detection. The precision was ca. 6% at a concentration of 300 ng/ml. Acid anhydrides were investigated as alternative reagents in the two-phase derivatization procedure, and heptafluorobutyric acid anhydride in aqueous solutions gave approximately 100% recovery. However, in urine the recoveries of the investigated acid anhydride derivatives were unsatisfactory.     

Stereochemical analysis of 3,4-methylenedioxymethamphetamine and its main metabolites by gas chromatography/mass spectrometry

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is consumed as the racemate but some metabolic steps are enantioselective. In addition, chiral properties are preserved during MDMA biotransformation. A quantitative analytical methodology using gas chromatography/mass spectrometry (GC/MS) to determine enantioselective disposition in the body of MDMA and its main metabolites including 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) was developed. Plasma and urine samples were collected from a male volunteer. The analysis of MDMA, MDA, and 4-hydroxy-3-methoxy metabolites by GC/MS required a two-step derivatization procedure. The first step consisted of derivatization of the amine with enantiomerically pure Mosher's reagent ((R)-MTPCl). Triethylamine was used as a base to neutralize hydrochloric acid formed during the reaction allowing quantitative derivatization, which resulted in a substantial improvement in the sensitivity of the method compared with other previously described techniques. Further treatment with ammonium hydroxide was required since both amine and hydroxyl groups underwent derivatization in the reaction. Ammonium hydroxide breaks bonds formed with hydroxyl groups without affecting amine derivatives. The second derivatization step using hexamethyldisilazane was needed for metabolites containing phenol residues. This derivatization method permitted the stereochemically specific study of MDMA and its main monohydroxylated metabolites by GC/MS. A detailed study of the chemical reactions involved in the derivatization steps was indispensable to develop a straightforward, sensitive, and reproducible method for the analysis of the parent drug compound and its metabolites.     

Determination of bisphenol A in foods as 2,2-bis-(4-(isopropoxycarbonyloxy)phenyl)propane by gas chromatography/mass spectrometry

A procedure has been developed for the determination of bisphenol A (BPA) in foods by gas chromatography/mass spectrometry as 2,2-bis-(4-(isopropoxycarbonyloxy)phenyl)propane formed in the reaction with isopropyl chloroformate. Optimal conditions have been found for BPA derivatization, providing its quantitative conversion into diether derivative in aqueous media. The concentration of BPA has been determined in some samples of canned foods and beverages (from 2.15 to 42.91 ng/g). The detection limit is 0.05–0.1 ng/g.     

The quantitative analysis of 1-alpha-acetylmethadol and its principal metabolites in biological specimens by gas chromatography-chemical ionization-multiple ion monitoring mass spectrometry.

1-alpha-acetylmethadol (LAAM) is a new drug under development for the treatment of heroin dependence. A new analytical method applicable to the accurate biodispositional study of the drug and its metabolities is described and critically discussed in this report. The procedure involves sample preparation and direct organic solvent extraction using eta-butyl chloride, amide derivatization by molecular rearrangement, and gas chromatography-chemical ionization mass spectrometry-selected ion monitoring, with methane as the carrier and ammonia as reagent gases. Deuterated (d3 stable isotopes of LAAM and its metabolites are used as internal standards. The method is free from qualitative interferences and has quantitative sensitivity to 5 ng/ml for 2.0 ml samples with 10-15% accuracy and precision in the range 5-100 ng/ml; and 2-5% at concentrations up to 750 ng/ml. Specimens of plasma, whole blood, urine, bile, brain, liver, and other visceral samples have been successfully analyzed, as well as in vitro preparations such as hepatic microsomes. By appropriate data processing, the method lends itself to routine analysis and high volume work; even manually the method is capable of at least 50 samples per week. A simplified procedure for the analysis of LAAM and its metabolites in urine only is also presented and discuet up and use the methods.     

超高效液相色谱-串联质谱法定性和高效液相色谱法定量分析天南星中氨基酸成分

为建立中药材中氨基类极性非紫外活性成分的定性与定量分析方法,以中药材天南星为研究对象,采用柱前衍生化技术,以异硫氰酸苯酯(PITC)为衍生化试剂,经C₁₈色谱柱(100 mm×2.1 mm, 3.5 μm)分离和超高效液相色谱-串联质谱(UHPLC-MS/MS)分析,共解析了天南星中20个成分,包括18个氨基酸和2个胺类化合物。经优化衍生化条件,应用高效液相色谱法(HPLC),以Diamonsil C₁₈色谱柱(250 mm×4.6 mm, 5 μm)分离,以乙腈和0.05 mol/L醋酸铵-醋酸缓冲液(pH 6.5)为流动相,梯度洗脱,在254 nm下检测,建立了同时测定15种氨基酸含量的方法,经方法学考察符合含量测定要求。谷氨酸、色氨酸在2~100 mg/L范围内、精氨酸在6~300 mg/L范围内、其余各氨基酸在0.8~40 mg/L范围内均呈良好的线性关系,相关系数均不小于0.9995;平均回收率在95%~105%之间,RSD均小于3%;并成功应用于12批中药材的测定。本方法简便、灵敏、准确,具有可操作性,可用于快速鉴定中药中的氨基类成分以及进行含量测定。     

A new approach for generic screening and quantitation of potential genotoxic alkylation compounds by pre-column derivatization and LC-MS/MS analysis

A generic LC-MS/MS method was developed for the analysis of potentially genotoxic alkyl halides. A broad selection of alkyl halides were derivatized using 4-dimethylaminopyridine in acetonitrile. The reaction conditions for derivatization, i.e., solvent, reaction time, temperature and concentration of alkyl halide, active pharmaceutical ingredient (API), and reagent, were optimized for sensitivity and robustness. The interference of the matrix and the API and the presence of water on the derivatization reaction were investigated for a model drug product (paracetamol/caffeine tablets). Hydrophilic interaction liquid chromatography was used to allow a quantitative determination of the derivatives by tandem mass spectrometry. The derivatization reaction was shown to be selective for alkyl halides, although some reactivity was also observed for an aromatic sulfonate, which is also genotoxic. Even though differences in reaction efficiencies have been observed, the enhanced sensitivity obtained by the derivatization allows the majority of the alkyl halides to be detected by MS/MS at relevant levels for genotoxic impurity evaluation, i.e., 10 mg kg(-1). Another key advantage is that for the majority of derivatives, reagent-related fragments are produced, which allows low-level screening for alkyl halides. Highly specific MS detection can be performed using neutral loss and precursor ion scan experiments. The applicability of a generic screening method will make the genotox evaluation less dependent on the quality of assessments based on predictions only, and it will provide essential information during the development of new chemical entities. In addition to screening, target analysis in the low milligrams per kilogram range can be performed. A similar response of the derivatized compounds was obtained in the range of 1-100 mg kg(-1) with a reproducibility better than 10%, which is sufficient for the determination of alkyl halides in APIs and drug products.     

Dynamic ultrasound-assisted extraction of colistin from feeds with on-line pre-column derivatization and liquid chromatography-fluorimetric detection

A dynamic ultrasound-assisted extraction (UAE) method with on-line pre-column derivatization/high performance liquid chromatography (HPLC) and fluorimetric detection is proposed for the analysis of colistin in feed. A flow injection manifold is used for the development of the extraction and derivatization steps and for interfacing them with the separation/detection step, thus providing an on-line approach with the advantage of minimum sample handling. The derivatization was performed with ortho-phthaldialdehyde and 2-mercaptoethanol. The optimum conditions for colistin extraction and formation of the fluorescent derivative have been obtained by experimental design methodology. The use of a high-intensity probe sonication makes UAE an expeditious (7 min versus > 1 h) and efficient (93.1-98.2% versus 87.5-94% of recovery) alternative as compared with extraction using an ultrasonic bath. The within-laboratory reproducibility and repeatability, expressed as percentage of relative standard deviation, were 5.2 and 5.8, respectively.     

Precolumn derivatization of reducing carbohydrates with 4-(3-Methyl-5-oxo-2-pyrazolin-1-yl) benzoic acid. Study of reaction, high-performance liquid chromatographic separation and quantitative performance of method

Summary A simple method for quantitative determination of carbohydrates by reversed-phase, high-performance liquid chromatography after pre-column derivatization and UV detection has been developed. Reducing sugars are condensed with 4-(3-methyl-5-oxo-2-pyrazolin-l-yl) benzoic acid (PMPA) to yield UV adducts absorbing at 271 nm, which are resolved under typical reversed-phase conditions. After derivatization, excess PMPA is easily removed from the reaction mixture by precipitation with mineral acids at pH<4. The influence of experimental conditions on reaction yield, as well as chromatographic separation of derivatives, were investigated. The quantitative performance was evaluated by means of a protocol comprising replicate measurements at several analyte levels. The calibration curves obtained for 8 sugars showed excellent linearity over 10–5000 pmol. Limits of detection and quantification for several monosaccharides were ca. 10 and 50 picomoles, respectively. Optimized conditions were successfully used for quantitative determination of monosaccharides released after hydrolysis from fetuin, mucin, α₁-acid glycoprotein, ovalbumin and transferrin.     

Simple and highly sensitive determination of free fatty acids in human serum by high performance liquid chromatography with fluorescence detection.

A highly sensitive and simple reversed phase high performance liquid chromatographic (HPLC) method for the quantitative determination of free fatty acids in human serum is presented. The method is based on the direct derivatization of serum fatty acids with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37 degrees C. The resulting derivatives are separated within 75 min on a reversed phase column (YMC Pack C8) with a gradient elution of aqueous acetonitrile and detected fluorimetrically. The detection limits are 2.5-5 fmol in a 10 microL injection volume. The sensitivity permits precise determination of free fatty acids in 5 microL serum. The method is simple and is without the conventional liquid-liquid extraction steps of serum fatty acids.     

Determination of trace biogenic amines with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene derivatization using high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method based on chemical derivatization with fluorescence detection has been developed for analyzing biogenic amines in food and environmental samples. A BODIPY-based fluorescent reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su), was employed for the derivatization of these biogenic amines at 20 °C for 20 min in pH 7.20 borate buffer after careful investigation of the derivatization conditions including reagent concentration, buffer solution, reaction temperature and reaction time. Separation of biogenic amines with gradient elution was conducted on a C8 column with methanol-tetrahydrofuran-water as mobile phase. The detection limits were obtained in the range from 0.1 to 0.2 nM (signal-to-noise=3). This procedure has been validated using practical samples. The study results demonstrated a potential of employing high-performance liquid chromatography (HPLC) with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene labeling as a tool for quantitative analysis of biogenic amines involved in various matrices.     

基于巯基化衍生的气相色谱-质谱法测定有机相及水相中氯化氰

氯化氰(ClCN)是常用的化工中间体,也是《禁止化学武器公约》附表颁布的化学毒剂之一.采用传统的比色法或气相色谱法对ClCN进行分析时,稳定性差且检出限高.研究建立了有机相及水相中ClCN的巯基化衍生过程及气相色谱-质谱(GC-MS)的检测方法.经比较后选择1-丁基硫醇作为衍生试剂,有机相中ClCN的衍生条件为衍生温度...     

Derivatization followed by gas chromatography-mass spectrometry for quantification of ethyl carbamate in alcoholic beverages

A sensitive and rapid analytical methodology based on derivatization followed by gas chromatography-mass spectrometry (GC-MS) was developed for the quantitative determination of the toxic contaminant ethyl carbamate (EC, urethane, C(2)H(5)OCONH(2)) in alcoholic samples. EC was extracted using liquid-liquid extraction technique, and then silylated with bis-(trimethylsilyl)trifluoroacetamide, analysed finally by GC-MS. The isopropyl carbamate was used as the internal standard for quantitative analysis of EC in alcoholic samples. In this work, the sample extraction and derivatization reaction conditions were investigated, and the optimal extraction conditions obtained were: pH 9 and solvent of ethyl acetate, and the derivatization conditions were: derivatization reaction temperature of 80°C and time duration of 30 min. With the optimal conditions, the method validations were also studied. In the validation studies, EC exhibited good linearity with a regression coefficient of 0.9999. The limit of detection and limit of quantification were 0.30 and 5.0 μg/kg, respectively. The precision was less than 8.4%. Finally, the proposed technique was successfully applied to the analysis of EC in 35 kinds of alcoholic samples. The experimental results have demonstrated that the proposed technique is a fast, reliable and low-cost method for determination of EC in alcoholic samples.     

Study of different parameters affecting the derivatization of acidic herbicides with trimethylsulfonium hydroxide to make them suitable for gas chromatography analysis

In this study, an orthogonal array design was applied to know the way different parameters affected the derivatization of some herbicides that are commonly applied in the soils. Herbicides formulated as esters have been reported to rapidly hydrolyse, in contact with soil, to their corresponding acids and phenols. What involves is that both forms need to be monitored. Acidic herbicides and phenols cannot be detected by gas chromatography (GC) due to their polarity and low volatility that cause peak asymmetry. Therefore, masking of these polar groups by eliminating the active hydrogen atom with derivatization to their corresponding esters/ethers is needed in order to yield products that possess enhanced volatility and improved GC properties. A lot of derivatization reagents have been proposed but trimethylsulfonium hydroxide (TMSH) was selected due to its easy and quantitative formation of methyl esters/ethers. It was observed that the addition of TMSH promoted not only esterification of acids/phenols but trans-esterification of the original non-hydrolyzed remaining esters to their corresponding methyl ones. As a result, methyl esters/ethers were the final product of both reactions. Different parameters were studied in the statistical design for both TMSH promoted reactions: type of solvent, pH, temperature and time of incubation. The amount of derivatization reagent was calculated to be high enough to ensure the complete derivatization of all compounds present in the sample. The reaction medium was shown as an important factor. The formation of some methyl esters/ethers decreased with increasing time and temperature because trans-esterification, being an equilibrium where the formation of smaller structures is promoted, was not enough shifted. However, the statistical analysis revealed that only the pH of the solution played an important role during the derivatization process. The presence of the anionic form of the acids appeared to be essential for derivatization, being diminished in strong acidic conditions. In addition, pre-heating was shown not to improve derivatization reaction, being easily carried on in the injector port of the GC system.     

Chemically removable derivatization reagent for liquid chromatography I. 2-(N-Phthalimido)ethyl 2-(dimethylamino)ethanesulfonate

A sulfonate derivatization reagent, 2-(N-phthalimido)ethyl 2-(dimethylamino)ethanesulfonate, was synthesized and examined for use in liquid chromatography. The reagent contains two key moieties, a chromophore (phthalimido) necessary for detection and a dimethylamino function that is chemically removable after derivatization. The reagent was applied to the derivatization of 2,4,6-trichlorophenol as a model analyte. The results indicated that the reagent can be readily removed after derivatization by simple acid treatment.     

A sensitivity enhanced high-performance liquid chromatography fluorescence method for the detection of nephrotoxic and carcinogenic aristolochic acid in herbal medicines

A new, sensitive and selective HPLC method with fluorescence detector (HPLC-FLD) for the determination of nephrotoxic and carcinogenic aristolochic acid (AA) in herbal medicines by using pre-column derivatization with zinc powder in acetic acid is presented. Variables governing the derivatization reaction, such as the amount of zinc powder and acetic acid, as well as the derivatization time were studied and optimized. An extended linear dynamic range over three orders of magnitude was observed for AA-I and AA-II (R(2)>0.9998). Method accuracy at low, medium and high spiked AA levels determined by the percentage mean deviation was below 4.4% and 7.2% for AA-I and AA-II, respectively. The detection limits of 0.39 ng/mL (AA-I) and 0.52 ng/mL (AA-II) were 2 orders of magnitude lower than those obtained from HPLC-MS or CE-ECD analyses, 3-4 orders of magnitude lower than those from HPLC-UV or CE-UV methods. The developed method has been applied for the determination of AA in herbal medicines. Among the tested samples, Guanmutong had the highest AA concentration (2607.0 microg/g AA-I, 711.2 microg/g AA-II). Comparison studies between HPLC-FLD and HPLC-MS/MS demonstrated that the two methods gave similar quantitative results for the selected herb samples.     

Simple and sensitive determination of citrinin in Monascus by GC-selected ion monitoring mass spectrometry.

A new method for the qualitative and quantitative analysis of citrinin in Monascus by gas-chromatography-selected ion monitoring (SIM) mass spectrometry has been developed. GC separation of citrinin in Monascus extract was achieved without the need for chemical derivatization, and could be detected as a single peak when the SIM mode selected 5 prominent fragmentations (m/z of 220, 205, 177, 105 and 91). The quantitative detection limit for citrinin was approximately 1 ppb. Finally, the GC-separated analyte from Monascus extract, at a retention time of 10.89 min, was examined by the method of pattern recognition by comparison with a citrinin standard. The results show that the 2 compounds had a 94% similarity when the SIM mode was used.     

Supercritical fluid extraction of chlorpyrifos and 3,5,6-trichloro-2-pyridinol from garden compost

A method was developed for the simultaneous supercritical carbon dioxide extraction of chlorpyrifos and its primary degradate, 3,5,6-trichloro-2-pyridinol (TCP), from garden compost. In situ derivatization with N,O-bis(trimethylsilyl)trifluoracetamide was necessary for extraction of TCP. Recoveries for TCP and chlorpyrifos were quantitative for spiked compost samples. Sodium chloride was used as the packing material in extractions with in situ derivatization. Optimum results were obtained for air-dried samples containing 4-7% moisture. No sample cleanup was required prior to analysis by GC-flame ionization detection. The effects of compost moisture content and ageing were investigated for chlorpyrifos recovery. No significantly negative effect on recovery for up to 20% (w/w) moisture for chlorpyrifos was observed. Effects of ageing showed a decrease in extraction efficiency over time with 52% recovery after 10 days.     

HPLC-UV method development and validation for the determination of low level formaldehyde in a drug substance

A reversed-phase high-performance liquid chromatographic method (HPLC) with diode-array detection is developed and validated for the quantitative determination of formaldehyde in a drug substance. Formaldehyde (HCHO) is reacted with 2,4-dinitrophenylhydrazine (DNPH) to form a Schiff base (HCHO-DNPH derivatization product), which has an absorbing maximum (lambda max) at 360 nm. The HPLC method employs a C8, 3-microm particle size analytical column (150 mm x 4.6 mm), 15-microL injection volume, column temperature controlled at 30 degrees C, detection at 360 nm, and a water-acetonitrile (55:45, v/v) mobile phase at a flow rate of 1 mL/min. These conditions resolve the HCHO-DNPH product from unreacted DNPH, the drug substance and related impurities, as well as diluent peaks within 20 min. The retention time of the HCHO-DNPH product is approximately 6.4 min. The method is linear, accurate in the specified range (0.33-333 ppm), and robust based on analyte (HCHO-DNPH derivatization product) stability in standard and sample. Detection limit is 0.03 ng (0.1 ppm).     

Coupling of sequential injection analysis and capillary electrophoresis - Laser-induced fluorescence via a valve interface for on-line derivatization and analysis of amino acids and peptides

The on-line coupling of sequential injection analysis (SIA) and capillary electrophoresis (CE) via an in-line injection valve is presented. The SIA system is used for automated derivatization of amino acids and peptides. Dichlorotriazinylaminofluorescein serves as the derivatization agent, thus enabling sensitive laser-induced fluorescence detection of the derivatized analytes. The SIA procedure includes the following steps: (a) introduction of reagent and sample zones in a holding coil, (b) sample and reagent mixing in a reaction coil, (c) stop-flow step for increase of the reaction time, and (d) delivery of derivatized sample into the loop of the micro-valve interface. A small portion of the analyte zone is introduced electrokinetically in the separation capillary via the valve interface and CE analysis is performed. Factors affecting the CE separation, such as pH, the borate and sodium dodecyl sulphate concentration of the background electrolyte have been optimized. The derivatization conditions have been studied to obtain a high reaction yield in a relative short time. The transfer of a part of the reaction plug into the loop of the valve interface has been optimized. Using des-Tyr(1)-[Met]-enkephalinamide as test compound, it is demonstrated that after automated derivatization, on-line electrophoretic analysis could be achieved. Glycine has been selected as the internal standard in order to correct for variations in reaction time and filling of the injection loop. For the enkephalin, good reproducibility (RSD<4.5% calculated by the ratio of the peak areas) and linearity (0.5-5 microg mL(-1), R(2)>or=0.994) are obtained with a detection limit of 30 ng mL(-1) (S/N=3).