In situ derivatization–liquid liquid extraction as a sample preparation strategy for the determination of urinary biomarker prolyl‐4‐hydroxyproline by liquid chromatography–tandem mass spectrometry

Polar analytes that possess protic functional groups have often been treated with alkyl chloroformates to decrease their polarity and increase their volatility prior to gas chromatography–mass spectrometry analysis. This derivatization reaction has two distinct advantages. It proceeds smoothly in aqueous media, and the desired reaction products are efficiently separated from interfering ionic components by their extraction into a water‐immiscible organic phase. In the present work, the derivatization–liquid liquid sample preparation was examined in detail for analysis of a potential urinary dipeptide biomarker l ‐prolyl‐4‐l ‐hydroxyproline (PHP) by downstream liquid chromatography coupled to electrospray mass spectrometry. PHP was treated with a series of alkyl and fluoroalkyl chloroformates in aqueous media, and the detected reaction products were investigated. Smooth conversion of PHP into the N‐isobutyloxycarbonyl isobutyl ester was accomplished by the coupled action of isobutanol, isobutyl chloroformate and the pyridine catalyst. This derivative afforded a highest detector response from all the derivatized forms examined, including the nonderivatized PHP. A simple isocratic elution on a common RP‐C18 HPLC column coupled with tandem mass spectrometry, and use of the synthesized heptadeuterated analog (D7‐PHP) as an internal standard, enabled validation of the method and determination of PHP in human urine in less than 5 min. The in situ derivatization–liquid liquid extraction has thus been demonstrated to be a useful sample preparation strategy for the analysis of polar metabolites by liquid chromatography–tandem mass spectrometry in the complex urine matrix. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of herbicides in environmental water samples by simultaneous in‐syringe magnetic stirring‐assisted dispersive liquid–liquid microextraction and silylation followed by GC–MS

An on‐line, fast, simple, selective, and sensitive method has been developed for the determination of three herbicides belonging to the following families: triazines (atrazine), chloroacetamide (alachlor), and phenoxy (2,4‐dichlorophenoxyacetic acid) in water samples. The method involves an in‐syringe magnetic stirring‐assisted dispersive liquid–liquid microextraction along with simultaneous silylation prior to their determination by gas chromatography with mass spectrometry. Extraction, derivatization, and preconcentration have been simultaneously performed using acetone as dispersive solvent, N‐methyl‐N‐tert‐butyldimethylsilyltrifluoroacetamide as derivatization agent and trichloroethylene as extraction solvent. After stirring for 180 s, the sedimented phase was transferred to a rotary micro‐volume injection valve (3 μL) and introduced by an air stream into gas chromatograph with mass spectrometry detector. Recovery and enrichment factors were 87.2–111.2% and 7.4–10.4, respectively. Relative standard deviations were in the ranges of 6.6–7.4 for intraday and 9.2–9.6 for interday precision. The detection limits were in the range of 0.045–0.03 μg/L, and good linearity was observed up to 200 μg/L, with R² ranging between 0.9905 and 0.9964. The developed method was satisfactorily applied to assess the occurrence of the studied herbicides in groundwater samples. The recovery test was also performed with values between 77 and 117%.     

An efficient biosorption‐based dispersive liquid‐liquid microextraction with extractant removal by magnetic nanoparticles for quantification of bisphenol A in water samples by gas chromatography‐mass spectrometry detection

In this work, a simple, fast, sensitive, and environmentally friendly method was developed for preconcentration and quantitative measurement of bisphenol A in water samples using gas chromatography with mass spectrometry. The preconcentration approach, namely biosorption‐based dispersive liquid‐liquid microextraction with extractant removal by magnetic nanoparticles was performed based on the formation of microdroplet of rhamnolipid biosurfactant throughout the aqueous samples, which accelerates the mass transfer process between the extraction solvent and sample solution. The process is then followed by the application of magnetic nanoparticles for easy retrieval of the analyte‐containing extraction solvent. Several important variables were optimized comprehensively including type of disperser solvent and desorption solvent, rhamnolipid concentration, volume of disperser solvent, amount of magnetic nanoparticles, extraction time, desorption time, ionic strength, and sample pH. Under the optimized microextraction and gas chromatography with mass spectrometry conditions, the method demonstrated good linearity over the range of 0.5–500 µg/L with a coefficient of determination of R² = 0.9904, low limit of detection (0.15 µg/L) and limit of quantification (0.50 µg/L) of bisphenol A, good analyte recoveries (84–120%) and acceptable relative standard deviation (1.8–14.9%, n = 6). The proposed method was successfully applied to three environmental water samples, and bisphenol A was detected in all samples.     

Accurate and simple determination of oxcarbazepine in human plasma and urine samples using switchable-hydrophilicity solvent in GC–MS

This work presents a sensitive and rapid analytical method for the determination of oxcarbazepine in human plasma and urine samples. A vortex-assisted switchable hydrophilicity solvent-based liquid phase microextraction (VA–SHS–LPME) was used to preconcentrate oxcarbazepine from the samples before the determination by gas chromatography mass spectrometry. The switchable hydrophilicity solvent was synthesized by protonating N,N-dimethylbenzylamine with carbon dioxide to make it totally miscible with an equivalent volume of water. Parameters of the VA–SHS–LPME method including volume of switchable hydrophilicity solvent, concentration/volume of sodium hydroxide and vortex period were systematically optimized. Under the optimum conditions, good linearity ranging from 27.03 to 353.47 μg/kg was obtained for the analyte. Limit of detection and quantitation values were found to be 6.2 and 21 μg/kg (mass base), respectively. The relative standard deviation was calculated as 6.9% for six replicate measurements of the lowest concentration of the calibration plot. Satisfactory recovery results were calculated in the range of 97–100% for human plasma and urine samples spiked at five different concentrations.     

Solvent‐vapor‐assisted liquid–liquid microextraction: A novel method for the determination of phthalate esters in aqueous samples using GC–MS

A novel liquid–liquid microextraction method, namely, solvent‐vapor‐assisted liquid–liquid microextraction for the determination of dimethyl phthalate, diethyl phthalate, dibutyl phthalate and bis(2‐ethylhexyl) phthalate in the aqueous samples using gas chromatography with mass spectrometry was developed. In the proposed method, extracting solvent was heated, and solvent vapor as the extracting phase was injected into the sample solution. As a result of the low temperature of the sample solution and higher density of the extracting phase than the aqueous medium, solvent vapor was condensed and an organic‐phase drop formed in the bottom of sample tube. Because of the gas status of the extracting solvent, the surface area between the extracting solvent and the aqueous sample was remarkably high. Under the optimized conditions, tetrachloride carbon was used as an extracting solvent. The method shows high coefficient of determination (R ²) values in the range of 0.5–200 and 1.0–200 ng/mL for the target analytes. Enrichment factors and limits of detection for the studied phthalates are obtained in the ranges of 2800–3000 and 0.15–0.3 ng/mL, respectively. Recoveries and relative standard deviations were in the range of 80.0–100.0 and 2.2–7.8%, respectively. The proposed method successfully used for analysis of several aqueous samples.     

Environmentally friendly solid‐phase microextraction coupled with gas chromatography and mass spectrometry for the determination of biogenic amines in fish samples

In this work, a facile and environmentally friendly solid‐phase microextraction assay based on on‐fiber derivatization coupled with gas chromatography and mass spectrometry was developed for determining four nonvolatile index biogenic amines (putrescine, cadaverine, histamine, and tyramine) in fish samples. In the assay, the fiber was firstly dipped into a solution with isobutyl chloroformate as derivatization reagent and isooctane as extraction solvent. Thus, a thin organic liquid membrane coating was developed. Then the modified fiber was immersed into sample solution to extract four important bioamines. Afterwards, the fiber was directly inserted into gas chromatography injection port for thermal desorption. 1,7‐Diaminoheptane was employed as internal standard reagent for quantification of the targets. The limits of detection of the method were 2.98–45.3 μg/kg. The proposed method was successfully applied to the detection of bioamines in several fish samples with recoveries ranging 78.9–110%. The organic reagent used for extraction was as few as microliter that can greatly reduce the harm to manipulator and environment. Moreover, the extraction procedures were very simple without concentration and elution procedures, which can greatly simplify the pretreatment process. The assay can be extended to the in situ screening of other pollutant in food safety by changing the derivatization reagent.     

Switchable‐hydrophilicity solvent‐based microextraction combined with gas chromatography for the determination of nitroaromatic compounds in water samples

A simple, fast, sensitive, and low‐cost method was developed for the quantification of nitroaromatic compounds in water samples based on CO₂‐assisted liquid‐phase microextraction using a switchable‐hydrophilicity solvent followed by gas chromatography detection. Dipropylamine was used as extraction solvent with switchable hydrophilicity that can be miscible or immiscible upon the addition or removal of CO₂ as a reagent. Experimental parameters affecting the extraction efficiency such as the volume of acceptor phase, the volume of donor phase, pH of donor phase, ionic strength, and extraction time were investigated. Under the optimal conditions, detection limits and preconcentration factors were obtained in the ranges of 0.9–1.8 μg/L and 132–138, respectively. Also, the extraction recoveries of water samples were above 88%. Finally, the developed method was successfully applied to the determination of nitroaromatic compounds in real water samples.     

A rapid method for the quantification of urinary phthalate monoesters: A new strategy for the assessment of the exposure to phthalate ester by solid‐phase microextraction with gas chromatography and tandem mass spectrometry

In the following work, a new method for the analysis of the phthalate monoesters in human urine was reported. Phthalate monoesters are metabolites generated as a result of phthalate exposure. In compliance with the dictates of Green Analytical Chemistry, a rapid and simple protocol was developed and optimized for the quantification of phthalate monoesters (i.e., monoethyl phthalate, monoisobutyl phthalate, mono‐n‐butyl phthalate, mono‐(2‐ethylhexyl) phthalate, mono‐n‐octyl phthalate, monocyclohexyl phthalate, mono‐isononyl phthalate) in human urine, which entails preceding derivatization with methyl chloroformate combined with the use of commercial solid phase microextraction and the analysis by gas chromatography‐triple quadrupole mass spectrometry. The affinity of the derivatized analytes toward five commercial coatings was evaluated, and in terms of analyte extraction, the best results were reached with the use of the divinylbenzene/carboxen/polydimethylsiloxane fiber. The multivariate approach of experimental design was used to seek for the best working conditions of the derivatization reaction and the solid phase microextraction, thus obtaining the optimum response values. The proposed method was validated according to the guidelines issued by the Food and Drug Administration achieving satisfactory values in terms of linearity, sensitivity, matrix effect, intra‐ and inter‐day accuracy, and precision.     

Determination of atenolol in human plasma using ionic‐liquid‐based ultrasound‐assisted in situ solvent formation microextraction followed by high‐performance liquid chromatography

An efficient analytical method called ionic‐liquid‐based ultrasound‐assisted in situ solvent formation microextraction followed by high‐performance liquid chromatography was developed for the determination of atenolol in human plasma. A hydrophobic ionic liquid (1‐butyl‐3‐methylimidazolium hexafluorophosphate) was formed by the addition of a hydrophilic ionic liquid (1‐butyl‐3‐methylimidazolium tetrafluoroborate) to a sample solution containing an ion‐pairing agent during microextraction. The analyte was extracted into the ionic liquid phase while the microextraction solvent was dispersed throughout the sample by utilizing ultrasound. The sample was then centrifuged, and the extracting phase retracted into the microsyringe and injected to liquid chromatography. After optimization, the calibration curve showed linearity in the range of 2–750 ng/mL with the regression coefficient corresponding to 0.998. The limits of detection (S/N = 3) and quantification (S/N = 10) were 0.5 and 2 ng/mL, respectively. A reasonable relative recovery range of 90–96.7% and satisfactory intra‐assay (4.8–5.1%, n = 6) and interassay (5.0–5.6%, n = 9) precision along with a substantial sample clean‐up demonstrated good performance of the procedure. It was applied for the determination of atenolol in human plasma after oral administration and some pharmacokinetic data were obtained.     

A novel and rapid extraction protocol for sensitive and accurate determination of prochloraz in orange juice samples: Vortex‐assisted spraying‐based fine droplet formation liquid‐phase microextraction before gas chromatography–mass spectrometry

A novel, ecofriendly, and easy extraction and preconcentration method named as vortex‐assisted spraying‐based fine droplet formation liquid‐phase microextraction was proposed for the determination of prochloraz at trace levels in orange juice samples by gas chromatography–mass spectrometry (GC‐MS). In this novel system, extraction solvent is dispersed by the help of spraying apparatus instead of dispersive solvent. Various parameters of the method were carefully optimized to increase signal‐to‐noise ratio of the analyte. Under the optimum chromatographic and extraction conditions, limit of detection and limit of quantification were calculated as 3.2 and 10.8 μg/kg, respectively. Moreover, enhancement in quantification power for the GC‐MS system was determined as 372 folds based on LOQ comparison. Relative recovery results for orange juice samples were found to be between 95.0–107.7% by utilizing matrix matching calibration. Furthermore, the developed method may be used to efficiently and simply extract other organic compounds for their determinations in several matrices.     

Use of switchable hydrophilicity solvents for the homogeneous liquid–liquid microextraction of triazine herbicides from environmental water samples

A homogeneous liquid–liquid microextraction alternative, based on the use of switchable hydrophilicity solvents, is presented. The extraction technique makes use of 125 μL of a water‐immiscible solvent (N,N‐dimethylcyclohexylamine) that can be solubilized in the aqueous phase in 1:1 ratio using CO₂ as a reagent. After the extraction, phase separation is induced by the addition of sodium hydroxide that produces a change on the ionization state of the amine, and centrifugation was not necessary. The extraction technique has been optimized and characterized using the determination of triazine herbicides by gas chromatography with mass spectrometry in water samples. The presence of metallic ions in environmental waters as interferents is easily avoided by the addition of ethylenediaminetetraacetic acid before the microextraction procedure. The proposed method allows the determination of the target analytes at the low microgram per liter range with good precision (relative standard deviation lower than 12.5%).     

Vortex‐assisted surfactant‐enhanced‐emulsification liquid–liquid microextraction of biogenic amines in fermented foods before their simultaneous analysis by high‐performance liquid chromatography

A simple, rapid, sensitive, and environmentally friendly method, based on modified dispersive liquid–liquid microextraction coupled with high‐performance liquid chromatography was developed for the simultaneous determination of five biogenic amines in fermented food samples. Biogenic amines were derivatized with 9‐fluorenylmethyl chloroformate, extracted by vortex‐assisted surfactant‐enhanced emulsification liquid–liquid microextraction, and then analyzed by high‐performance liquid chromatography. Five biogenic amine compounds were separated within 30 min using a C₁₈ column and gradient elution with acetonitrile and 1% acetic acid. Factors influencing the derivatization and extraction efficiency such as type and volume of extraction solvent, type, and concentration of surfactant, pH, salt addition, and vortex time were optimized. Under the optimum conditions, the method provided the enrichment factors in the range of 161–553. Good linearity was obtained from 0.002–0.5 mg/L for cadaverine and tyramine, 0.003–1 mg/L for tryptamine and histamine, and 0.005–1 mg/L for spermidine with coefficient of determination (R²) > 0.992. The limits of detection ranged from 0.0010 to 0.0026 mg/L. The proposed method was successfully applied to analysis of biogenic amines in fermented foods such as fermented fish (plaa‐som), wine and beer where good recoveries were obtained in the range of 83.2–112.5%     

Rapid and sensitive determination of phytosterols in functional foods and medicinal herbs by using UHPLC–MS/MS with microwave‐assisted derivatization combined with dual ultrasound‐assisted dispersive liquid–liquid microextraction

In this work, a hyphenated technique of dual ultrasound‐assisted dispersive liquid–liquid microextraction combined with microwave‐assisted derivatization followed by ultra high performance liquid chromatography tandem mass spectrometry has been developed for the determination of phytosterols in functional foods and medicinal herbs. Multiple reaction monitoring mode was used for the tandem mass spectrometry detection. A mass spectrometry sensitive reagent, 4′‐carboxy‐substituted rosamine, has been used as the derivatization reagent for five phytosterols, and internal standard diosgenin was used for the first time. Parameters for the dual microextraction, microwave‐assisted derivatization, and ultra high performance liquid chromatography tandem mass spectrometry were all optimized in detail. Satisfactory linearity, recovery, repeatability, accuracy and precision, absence of matrix effect, extremely low limits of detection (0.005–0.015 ng/mL) and limits of quantification (0.030–0.10 ng/mL) were achieved. The proposed method was compared with previously reported methods. It showed better sensitivity, selectivity, and accuracy. The matrix effect was also significantly reduced. The proposed method was successfully applied to the determination of five phytosterols in vegetable oil (sunflower oil, olive oil, corn oil, peanut oil), milk and orange juice (soymilk, peanut milk, orange juice), and medicinal herbs (Ginseng, Ganoderma lucidum, Cordyceps, Polygonum multiflorum) for the quality control of functional foods and medicinal herbs.     

Automated headspace solid‐phase microextraction and on‐fiber derivatization for the determination of clenbuterol in meat products by gas chromatography coupled to mass spectrometry

A method was developed for the determination of clenbuterol in meat using stable‐isotope‐dilution gas chromatography with mass spectrometry coupled with solid‐phase microextraction and on‐fiber derivatization. The samples were first homogenized with hydrochloric acid followed by protein deposition. After headspace solid‐phase microextraction and on‐fiber derivatization, the content of clenbuterol was measured with the aid of stable‐isotope dilution. The condition of solid‐phase microextraction was optimized by central composite design. The relative standard deviations, limit of detection, and recoveries for clenbuterol were 4.2–9.2%, 0.48 μg/kg, and 96–104%, respectively. The proposed method was satisfactory for analysis of real samples as compared with the Chinese standard method.     

Suitability of dispersive liquid–liquid microextraction for the in situ silylation of chlorophenols in water samples before gas chromatography with mass spectrometry

Trace analysis of chlorophenols in water was performed by simultaneous silylation and dispersive liquid–liquid microextraction followed by gas chromatography with mass spectrometry. Dispersive liquid–liquid microextraction was carried out using an organic solvent lighter than water (n‐hexane). The effect of different silylating reagents on the method efficiency was investigated. The influence of derivatization reagent volume, presence of catalyst and derivatization/extraction time on the yield of the derivatization reaction was studied. Different parameters affecting extraction efficiency such as kind and volume of extraction and disperser solvents, pH of the sample and addition of salt were also investigated and optimized. Under the optimum conditions, the calibration graphs were linear in the range of 0.05–100 ng/mL and the limit of detection was 0.01 ng/mL. The enrichment factors were 242, 351, and 363 for 4‐chlorophenol, 2,4‐dichlorophenol, and 2,4,6‐trichlorophenol, respectively. The values of intra‐ and inter‐day relative standard deviations were in the range of 3.0–6.4 and 6.1–9.9%, respectively. The applicability of the method was investigated by analyzing water and wastewater samples.     

Determination of desipramine in biological samples using liquid–liquid–liquid microextraction combined with in‐syringe derivatization,gas chromatography,and nitrogen/phosphorus detection

A method was established for the determination of desipramine in biological samples using liquid–liquid–liquid microextraction followed by in‐syringe derivatization and gas chromatography–nitrogen phosphorus detection. The extraction method was based on the use of two immiscible organic solvents. n‐Dodecane was impregnated in the pores of the hollow fiber and methanol was placed inside the lumen of the fiber as the acceptor phase. Acetic anhydride was used as the reagent for the derivatization of the analyte inside the syringe barrel. Parameters that affect the extraction efficiency (composition of donor and acceptor phase, ionic strength, sample temperature, and extraction time) as well as derivatization efficiency (amount of acetic anhydride and reaction time and temperature) were investigated. The limit of detection was 0.02 μg/L with intra and interday RSDs of 2.6 and 7.7%, respectively. The linearity of the method was in the range of 0.2–20 μg/L (r² = 0.9986). The method was successfully applied to determine desipramine in human plasma and urine.     

Switchable‐hydrophilicity solvent liquid‐liquid microextraction versus dispersive liquid‐liquid microextraction prior to HPLC‐UV for the determination and isolation of piperine from Piper nigrum L

Switchable‐hydrophilicity solvent liquid‐liquid microextraction and dispersive liquid‐liquid microextraction were compared for the extraction of piperine from Piper nigrum L. prior to its analysis by using high‐performance liquid chromatography with UV detection. Under optimum conditions, limits of detection and quantitation were found as 0.2–0.6 and 0.7–2.0 μg/mg with the two methods, respectively. Calibration graphs showed good linearity with coefficients of determination (R²) higher than 0.9962 and percentage relative standard deviations lower than 6.8%. Both methods were efficiently used for the extraction of piperine from black and white pepper samples from different origins and percentage relative recoveries ranged between 90.0 and 106.0%. The results showed that switchable‐hydrophilicity solvent liquid‐liquid microextraction is a better alternative to dispersive liquid‐liquid microextraction for the routine analysis of piperine in food samples. A novel scaled‐up dispersive liquid‐liquid microextraction method was also proposed for the isolation of piperine providing a yield of 102.9 ± 4.9% and purity higher than 98.0% as revealed by NMR spectroscopy.     

Trace analysis of rimantadine in human urine after dispersive liquid liquid microextraction followed by liquid chromatography–post column derivatization

The first dispersive liquid liquid microextraction scheme followed by liquid chromatography‐post column derivatization for the determination of the antiviral drug rimantadine in urine samples is demonstrated. The effect of the type and volume of organic extraction solvent, type and volume of disperser solvent, sample pH, ionic strength, extraction time, and centrifugation speed on the extraction efficiency were studied. Rimantadine and the internal standard (amantadine) were chromatographed using a reversed phase monolithic stationary phase with a mixture of equal volumes of methanol and phosphate buffer (pH = 3) as mobile phase. On‐line post‐column derivatization of the analyte was performed using a “two‐stream” manifold with o‐phthalaldehyde and N‐acetyl‐cysteine at alkaline medium. Under the optimized extraction conditions, the enrichment factor of rimantadine was 58. The linear range was 5–100 µg/L with correlation coefficient r of 0.9984 while the limit of detection achieved was 0.5 µg/L. The within‐day and between‐day precision for the tested concentration levels were less than 14.3% and the mean recoveries obtained from the spiked samples were ranged between 87.5 and 113.9%. The main advantages of the proposed method are the simplicity of operation, rapidity, low cost, and low limit of detection of the analyte.     

Matrix solid‐phase dispersion with chitosan‐zinc oxide nanoparticles combined with flotation‐assisted dispersive liquid–liquid microextraction for the determination of 13 n‐alkanes in soil samples

In this study, chitosan‐zinc oxide nanoparticles were used as a sorbent of miniaturized matrix solid‐phase dispersion combined with flotation‐assisted dispersive liquid–liquid microextraction for the simultaneous determination of 13 n‐alkanes such as C₈H₁₈ and C₂₀H₄₂ in soil samples. The solid samples were directly blended with the chitosan nanoparticles in the solid‐phase dispersion method. The eluent of solid‐phase dispersion was applied as the dispersive solvent for the following flotation‐assisted dispersive liquid–liquid microextraction for further purification and enrichment of the target compounds prior to gas chromatography with flame ionization detection. Under the optimum conditions, good linearity with correlation coefficients in the range 0.9991 < r² < 0.9995 and low detection limits between 0.08 to 2.5 ng/g were achieved. The presented procedure combined the advantages of chitosan‐zinc oxide nanoparticles, solid‐phase dispersion and flotation‐assisted dispersive liquid–liquid microextraction, and could be applied for the determination of n‐alkanes in complicated soil samples with acceptable recoveries.     

Magnetic solid‐phase extraction or dispersive liquid–liquid microextraction for pyrethroid determination in environmental samples

The determination of 15 pyrethroids in soil and water samples was carried out by gas chromatography with mass spectrometry. Compounds were extracted from the soil samples (4 g) using solid–liquid extraction and then salting‐out assisted liquid–liquid extraction. The acetonitrile phase obtained (0.8 mL) was used as a dispersant solvent, to which 75 μL of chloroform was added as an extractant solvent, submitting the mixture to dispersive liquid–liquid microextraction. For the analysis of water samples (40 mL), magnetic solid‐phase extraction was performed using nanocomposites of magnetic nanoparticles and multiwalled carbon nanotubes as sorbent material (10 mg). The mixture was shaken for 45 min at room temperature before separation with a magnet and desorption with 3 mL of acetone using ultrasounds for 5 min. The solvent was evaporated and reconstituted with 100 μL acetonitrile before injection. Matrix‐matched calibration is recommended for quantification of soil samples, while water samples can be quantified by standards calibration. The limits of detection were in the range of 0.03–0.5 ng/g (soil) and 0.09–0.24 ng/mL (water), depending on the analyte. The analyzed environmental samples did not contain the studied pyrethroids, at least above the corresponding limits of detection.