Assay of urinary 8‐hydroxy‐2′‐deoxyguanosine by capillary electrophoresis with spectrophotometric detection using a high‐sensitivity detection cell and solid‐phase extraction

A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z‐cell was devised for the assay of 8‐hydroxy‐2′‐deoxyguanosine (8OHdG) in human urine. Solid‐phase extraction (SPE) based on hydrophilic‐lipophilic‐balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused‐silica capillaries employing a Z‐cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10–1000 ng/mL; R² = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.     

Study on Determination of Six Transition Metal Ions in Biological Samples by SPE and RP‐HPLC

This paper reports the utilization of solid phase extraction and the reversed‐phase high‐performance liquid chromatography (RP‐HPLC) for the determination of six transition metal ions (iron, cobalt, nickel, copper, zinc and manganese) in biological samples. The samples were digested by microwave digestion. The iron, cobalt, nickel, copper, zinc and manganese ions in the digested samples can react with 2‐(2‐quinolinylazo)‐5‐diethylaminophenol (QADEAP) to form colored chelates in pH 4.0 acetic acid‐sodium acetic buffer solutions and cetyl trimethylammonium bromide (CTMAB) medium. These chelates were enriched by solid phase extraction with C₁₈ cartridge. Then the chelates were separated on a Waters Nova‐Pak‐C₁₈ column (3.9 × 150 mm, 5 μm) by gradient elution with methanol (containing 0.5% of acetic acid and 0.1% of CTMAB) and 0.05 mol/L pH 4.0 acetic acid‐sodium acetic buffer solution (containing 0.1% of CTMAB) as mobile phase at a flow rate of 0.5 mL/min. The detection limits of iron, cobalt, nickel, copper, zinc and manganese are 3 ng/L, 4 ng/L, 2 ng/L, 4 ng/L, 8 ng/L, 10 ng/L, respectively. This method was applied to the determination of iron, cobalt, nickel, copper, zinc and manganese in biological samples with good results.     

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.     

A CE‐LIF method based on long wavelength fluorescence labeling for the analysis of thiols in human urine

A rapid and robust CE method using a long wavelength fluorescent reagent 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(2‐maleimide)difluoroboradiaza‐s‐indacene as the labeling reagent has been developed for the simultaneous determination of thiols, including glutathione, cysteine, homocysteine, N‐acetylcysteine, cysteinylglycine, and penicillamine. The derivatization reaction was carried out in 14 mmol/L pH 8.5 borate buffer at 30°C for 6 min and the labeled thiols derivatives were separated with the running buffer containing 30 mmol/L pH 7.4 phosphate, 30% v/v acetonitrile and 8 mmol/L SDS within 12 min. Detection limits ranged from 0.4 to 2.4 nmol/L. To demonstrate the capability of this method, it was applied to the analysis of thiols in human urine with recoveries of 92.4–105.6%. The derivatization reaction was much faster at milder conditions, and the analysis was rapider. Moreover, with excitation wavelength at long wavelength region, background interference from samples was reduced effectively. The present method seems to be a potential choice for quantifying thiols in human urine.     

Eco‐friendly ionic liquid assisted capillary electrophoresis and α‐acid glycoprotein‐assisted liquid chromatography for simultaneous determination of anticancer drugs in human fluids

In the current work, two eco‐friendly analytical methods based on capillary electrophoresis (CE) and reversed phase liquid chromatography (RPLC) were developed for simultaneous determination of the most commonly used anticancer drugs for Hodgkin's disease: methotrexate (MTX), vinblastine, chlorambucil and dacarbazine. A background electrolyte (BGE) of 12.5 mmol/L phosphate buffer at pH 7.4 and 0.1 µmol/L 1‐butyl‐3‐methyl imidazolium bromide (BMImBr) ionic liquid (IL) was used for CE measurements at 250 nm detection wavelength, 20 kV applied voltage and 25 °C. The rinsing protocol was significantly improved to reduce the adsorption of IL on the interior surface of capillary. Moreover, RPLC method was developed on α‐1‐acid glycoprotein (AGP) column. Mobile phase was 10 mmol/L phosphate buffer at pH 6.0 (100% v/v) and flow rate at 0.1 mL/min. As AGP is a chiral column, it was successfully separated l ‐MTX from its enantiomer impurity d ‐MTX. Good linearity of quantitative analysis was achieved with coefficients of determinations (r²) >0.995. The stability of drugs measurements was investigated with adequate recoveries up to 24 h storage time under ambient temperature. The limits of detection were <50 and 90 ng/mL by CE and RPLC, respectively. The using of short‐chain IL as an additive in BGE achieved 600‐fold sensitivity enhancement compared with conventional Capillary Zone Electrophoresis (CZE). Therefore, for the first time, the proposed methods were successfully applied to determine simultaneously the analytes in human plasma and urine samples at clinically relevant concentrations with fast and simple pretreatments. Developed IL‐assisted CE and RPLC methods were also applied to measure MTX levels in patients’ samples over time. Copyright © 2014 John Wiley & Sons, Ltd.     

Resolution and isolation of enantiomers of (±)‐isoxsuprine using thin silica gel layers impregnated with l‐glutamic acid,comparison of separation of its diastereomers prepared with chiral derivatizing reagents having l‐amino acids as chiral auxiliaries

Thin silica gel layers impregnated with optically pure l ‐glutamic acid were used for direct resolution of enantiomers of (±)‐isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l ‐alanine, l ‐valine and S‐benzyl‐l ‐cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography on a C₁₈ column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin‐layer chromatography (TLC) on reversed phase (RP) C₁₈ plates. Diastereomers prepared with enantiomerically pure (+)‐isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)‐isoxsuprine. The elution order in the experimental study of RP‐TLC and RP‐HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1–0.09 µg/mL in TLC while it was in the range of 22–23 pg/mL in HPLC and 11–13 ng/mL in RP‐TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of galanthamine in Bulbus Lycoridis Radiatae by coupling capillary electrophoresis with end‐column electrochemiluminescence detection

A novel method for the determination of galanthamine (GAL) in Bulbus Lycoridis Radiatae has been developed based on coupling CE with an end‐column tris(2,2′‐bipyridyl)ruthenium(II) electrochemiluminescence (ECL). Parameters affecting CE separation and ECL detection were investigated and optimized. Baseline separation of GAL from other components in the Bulbus Lycoridis Radiatae sample was achieved with an 18 mmol/L phosphate running buffer at pH 9.0. Under the optimized conditions: 12 kV CE‐separation voltage, ECL detection potential at 1.25 V with 5 mmol/L and 50 mmol/L phosphate buffer at pH 7.5 in the detection reservoir, the linear range of GAL concentration was from 0.8 ng/mL to 2 μg/mL, whereas the detection limit was 0.25 ng/mL (S/N=3). The proposed method was successfully demonstrated for the determination of GAL in Bulbus Lycoridis Radiatae.     

Rapid determination of bisphenol A in drinking water using dispersive liquid‐phase microextraction with in situ derivatization prior to GC‐MS

This paper described a novel approach for the determination of bisphenol A by dispersive liquid‐phase microextraction with in situ acetylation prior to GC‐MS. In this derivatization/extraction method, 500 μL acetone (disperser solvent) containing 30.0 μL chlorobenzene (extraction solvent) and 30.0 μL acetic anhydride (derivatization reagent) was rapidly injected into 5.00 mL aqueous sample containing bisphenol A and K₂CO₃ (0.5% w/v). Within a few seconds the analyte was derivatized and extracted at the same time. After centrifugation, 1.0 μL of sedimented phase containing enriched analyte was determined by GC‐MS. Some important parameters, such as type and volume of extraction and disperser solvent, volume of acetic anhydride, derivatization and extraction time, amount of K₂CO₃, and salt addition were studied and optimized. Under the optimum conditions, the LOD and the LOQ were 0.01, 0.1 μg/L, respectively. The experimental results indicated that there was linearity over the range 0.1–50 μg/L with coefficient of correlation 0.9997, and good reproducibility with RSD 3.8% (n = 5). The proposed method has been applied for the analysis of drinking water samples, and satisfactory results were achieved.     

Affinity capillary electrophoresis coupling with partial filling technique and field‐amplified sample injection for enantioseparation and determination of DL‐tetrahydropalmatine

A novel, simple and sensitive method for the enantioseparation and determination of DL ‐tetrahydropalmatine (DL ‐THP) was developed using ACE in combination with partial filling technique and field‐amplified sample injection. A chiral selector, i.e. BSA, was used for the enantioseparation of DL ‐THP in ACE. Effects of BSA concentration, pH and separation voltage on the effectiveness of the enantiomer separation were evaluated. In an optimal condition, D ‐ and L ‐THP were completely enantio‐separated in less than 9 min by partially filling an electrophoretic capillary with 50 μmol/L BSA (50 mbar, 100 s) and carrying out an electrophoresis with 20 mmol/L phosphate buffer (pH 7.4) at 15 kV. The sensitivity was further improved by making use of field‐amplified sample injection to lower the LOD (defined as S/N=3) down to 6 ng/mL. Real samples were also tested and promising results for the determination of DL ‐THP enantiomers were obtained.     

An expedient reverse‐phase high‐performance chromatography (RP‐HPLC) based method for high‐throughput analysis of deferoxamine and ferrioxamine in urine

The present study was planned to optimize and validate an expedient reverse‐phase high chromatography (RP‐HPLC) based protocol for the analysis of deferoxamine (DFO) and ferrioxamine (FO) in urinary execration of patients suffering β ‐thalassemia major. The optimized RP‐HPLC method was found to be linear over the wide range of DFO and FO concentration (1–90 μg/mL) with appreciable recovery rates (79.64–97.30%) of quality controls at improved detection and quantitation limits and acceptable inter and intraday variability. Real‐time analysis of DFO and FO in the urine of thalassemic patients (male and female) at different intervals of Desferal®(Novartis Pharmaceuticals Corporation) injection revealed DFO and FO excretion at significantly (p < 0) different rates. The maximum concentrations of DFO (76.7 ± 3.06 μg/mL) and FO (74.2 ± 3.25 μg/mL) were found in urine samples, collected after 6 h of drug infusion while the minimum levels of DFO (1.10 ± 0.12 μg/mL) and FO (2.97 ± 0.13 μg/mL) were excreted by patients after 24 h. The present paper offers balanced conditions for an expedient, reliable and quick determination of DFO and FO in urine samples.     

Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous stereoselective analysis of naftopidil and O‐desmethyl naftopidil enantiomers in rat feces using an online column‐switching high‐performance liquid chromatography method

A novel online column‐switching chiral high‐performance liquid chromatography method was developed and validated for the simultaneous determination of naftopidil (NAF) and its O‐desmethyl metabolites (DMN) enantiomers in rat feces. Direct and multiple injections of supernatant from rat feces homogenate were allowed through the column‐switching system. Analyte extraction was performed on the Capcell Pak mixed‐functional column by acetonitrile–phosphate buffer (pH 7.4; 10 mm ; 8:92, v/v) flowing at 1 mL/min. Separation of NAF and DMN enantiomers was achieved on the Chiralpak IA column by methanol–acetonitrile–acetate buffer (pH 5.3; 5 mm ; 45:33:22, v/v/v) flowing at 0.5 mL/min. The analytes were measured with a fluorescence detector at 290 nm (λ_ex) and 340 nm (λ_em). The validated method showed a good linearity [22.5–15,000 ng/mL for (+)‐/(?)‐NAF; 35–25,000 ng/mL for (+)‐/(?)‐DMN] and the lowest limits of quantification for NAF and DMN enantiomers were 22.5 and 35 ng/mL, respectively. Both intra‐ and inter‐day variations were <10%. The assay was successfully applied to the fecal excretion of NAF and DMN enantiomers in rat after single oral administration of (±)‐NAF. Nonstereoselective excretion of (+)‐ and (?)‐NAF was found in feces, while stereoselective excretion of (+)‐ and (?)‐DMN was observed with higher excretion levels of (+)‐DMN, indicating that there may exist stereoselective metabolism for NAF enantiomers. Copyright © 2014 John Wiley & Sons, Ltd.     

Magnetic mixed hemimicelles solid‐phase extraction coupled with high‐performance liquid chromatography for the extraction and rapid determination of six fluoroquinolones in environmental water samples

In this study, a mixed hemimicelle solid‐phase extraction method based on Fe₃O₄ nanoparticles coated with sodium dodecyl sulfate was applied for the preconcentration and fast isolation of six fluoroquinolones in environmental water samples before high‐performance liquid chromatography determination. The main factors affecting the extraction efficiency of the analytes, such as amount of surfactant, amount of Fe₃O₄ nanoparticles, extraction time, sample volume, sample pH, ionic strength, and desorption conditions, were investigated and optimized. The method has detection limits from 0.05 to 0.1 ng/mL and good linearity (r ≥ 09948) in the range 0.1–200 ng/mL depending on the fluoroquinolone. The enrichment factor is ～200. The recoveries (at spiked levels of 1, 5, and 50 ng/mL) are in the range of 79–120%.     

Analysis of Phenylpropanolamine by Micellar Electroknetic Chromatography with Laser‐induced Fluorescence Detection

A new analytical method for phenylpropanolamine based on micellar electrokinetic chromatographic separation and laser‐induced fluorescence detection has been developed. Naphthalene‐2,3‐dicarboxaldehyde was used for precolumn derivatization of the nonfluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) containing 15 mM sodium dodecyl sulfate and a He‐Cd laser (Δ_ex: 442 nm, Δ_em: 500 nm). Linearity (r ≥ 0.99) of two orders of magnitude was generally obtained and the concentration limit of detection was in the ng/mL level. Coupled with a simple cleanup procedure, the method can be applied to the analysis of phenylpropanolamine in human plasma, with a limit of detection at 15 ng/mL. Recovery of phenylpropanolamine from plasma samples was about 90%.     

A novel fast method for aqueous derivatization of THC,OH‐THC and THC‐COOH in human whole blood and urine samples for routine forensic analyses

A novel aqueous in situ derivatization procedure with propyl chloroformate (PCF) for the simultaneous, quantitative analysis of Δ⁹‐tetrahydrocannabinol (THC), 11‐hydroxy‐Δ⁹‐tetrahydrocannabinol (OH‐THC) and 11‐nor‐Δ⁹‐tetrahydrocannabinol‐carboxylic acid (THC‐COOH) in human blood and urine is proposed. Unlike current methods based on the silylating agent [N,O‐bis(trimethylsilyl)trifluoroacetamide] added in an anhydrous environment, this new proposed method allows the addition of the derivatizing agent (propyl chloroformate, PCF) directly to the deproteinized blood and recovery of the derivatives by liquid–liquid extraction. This novel method can be also used for hydrolyzed urine samples. It is faster than the traditional method involving a derivatization with trimethyloxonium tetrafluoroborate. The analytes are separated, detected and quantified by gas chromatography–mass spectrometry in selected ion monitoring mode (SIM). The method was validated in terms of selectivity, capacity of identification, limits of detection (LOD) and quantification (LOQ), carryover, linearity, intra‐assay precision, inter‐assay precision and accuracy. The LOD and LOQ in hydrolyzed urine were 0.5 and 1.3 ng/mL for THC and 1.2 and 2.6 ng/mL for THC‐COOH, respectively. In blood, the LOD and LOQ were 0.2 and 0.5 ng/mL for THC, 0.2 and 0.6 ng/mL for OH‐THC, and 0.9 and 2.4 ng/mL for THC‐COOH, respectively. This method was applied to 35 urine samples and 50 blood samples resulting to be equivalent to the previously used ones with the advantage of a simpler method and faster sample processing time. We believe that this method will be a more convenient option for the routine analysis of cannabinoids in toxicological and forensic laboratories.     

Application of simultaneous separation and derivatization for the determination of α‐lipoic acid in urine samples by high‐performance liquid chromatography with spectrofluorimetric detection

To help to clarify therapeutic functions of lipoic acid (LA) in biochemical and clinical practice we have elaborated a fast, simple and accurate HPLC method enabling determination of LA in human urine. The proposed analytical approach includes reduction of LA with tris(2‐carboxyethyl)phosphine and simultaneous separation and derivatization of the analyte with butylamine and o‐phthaldialdehyde followed by spectrofluorimetric detection at λ_ex = 340 nm and λ_em = 440 nm. The assay was performed using gradient elution and the mobile phase containing 0.0025 mol L^?1 o‐phthaldialdehyde in 0.0025 mol L^?1 NaOH and acetonitrile. Linearity of the detector response for LA was observed in the range of 0.3–8 μmol L^?1. Limits of detection and quantification for LA in urine samples were 0.02 and 0.03 μmol L^?1, respectively. The total analysis time, including sample work‐up, was <20 min. The analytical procedure was successfully applied to analysis of real urine samples delivered from six healthy volunteers who received a single 100 mg dose of LA.     

Simultaneous determination of 9‐dehydro‐17‐hydro‐andrographolide and sodium 9‐dehydro‐17‐hydro‐andrographolide‐19‐yl sulfate in rat plasma by UHPLC‐ESI‐MS/MS after administration of xiyanping injection: application to a pharmacokinetic study

9‐Dehydro‐17‐hydro‐andrographolide (DHA) and sodium 9‐dehydro‐17‐hydro‐andrographolide‐19‐yl sulfate (DHAS) are active ingredients of xiyanping injection in clinical use. A simple, rapid and sensitive UHPLC‐ESI‐MS/MS method was developed for the determination of DHA and DHAS in rat plasma, and the pharmacokinetics of DHA and DHAS after intravenous administration of xiyanping injection was investigated. The plasma samples were treated with methanol to precipitate out protein, and the separation of DHA and DHAS was achieved on a Waters BEH C₁₈ column with a mobile phase consisting of acetonitrile and 10 mmol/L ammonium acetate solution at a flow rate of 0.4 mL/min. DHA, DHAS and the internal standard (internal standard, IS) diethylstilbestrol were detected at negative ion mode. The precursor‐product ion pairs used in multiple reaction monitoring mode were: m/z 349.1 → 286.9 (DHA), m/z 428.9 → 96.0 (DHAS) and m/z 267.1 → 236.9 (IS). Calibration curves offered satisfactory linearity within the test range, and all correlation coefficients were >0.995. The lower limit of detection of DHA and DHAS in plasma samples were determined to be 0.1 ng/mL. The lower limit of quantitation was 0.5 ng/mL for DHA and DHAS. All the recoveries of the quality control samples were in the range of 86.0–102.4%. The ratios of matrix effect were between 89.2 and 105.1%. The method was fully validated and successfully applied to the pharmacokinetic study of DHA and DHAS in rats. The study showed that both DHA and DHAS were distributed and eliminated rapidly in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

A new fluorescent derivatization reagent and its application to free fatty acid analysis in pomegranate samples using HPLC with fluorescence detection

A new fluorescent labeling reagent has been developed for the determination of fatty acids (FAs) by HPLC with fluorescence detection. The derivatization conditions including the amount of derivatization reagent, temperature, and type of catalyst were investigated, the results indicated that the reaction proceeded within 30 min at 90°C in the presence of K₂CO₃ catalyst. The maximal yield was obtained with a four‐ to fivefold molar reagent excess. The derivatives exhibited strong fluorescence with an excitation maximum at λ_ex = 245 nm and an emission maximum at λ_em = 410 nm. Twenty‐five FA derivatives were well separated by RP‐HPLC on a Hypersil BDS C₈ column in combination with gradient elution. All FAs were found to give excellent linear responses with correlation coefficients >0.9992. The method gave a low LOQ of 0.85–5.5 ng/mL (S/N of 10). The developed method was employed to analyze free FAs (FFAs) composition in pomegranate samples without any purification. FFAs in samples were doubly identified by HPLC retention time and protonated molecular ion corresponding to m/z [M+H]⁺. This newly developed method allows a highly sensitive determination of trace FFAs from pomegranate and other foodstuffs.     

Determination of trace hydroxyl polycyclic aromatic hydrocarbons in urine using graphene oxide incorporated monolith solid‐phase extraction coupled with LC‐MS/MS

The biomonitoring of hydroxy polycyclic aromatic hydrocarbons in urine, as a direct way to access multiple exposures to polycyclic aromatic hydrocarbons, has raised great concerns due to their increasing hazardous health effects on humans. Solid‐phase extraction is an effective and useful technique to preconcentrate trace analytes from biological samples. Here, we report a novel solid‐phase extraction method using a graphene oxide incorporated monolithic syringe for the determination of six hydroxy polycyclic aromatic hydrocarbons in urine coupled with liquid chromatography‐tandem mass spectrometry. The effect of graphene oxide amount, washing solvent, eluting solvent, and its volume on the extraction performance were investigated. The fabricated monoliths gave higher adsorption efficiency and capacity than the neat polymer monolith and commercial C₁₈ sorbent. Under the optimum conditions, the developed method provided the detection limits (S/N = 3) of 0.02–0.1 ng/mL and the linear ranges of 0.1–1500 ng/mL for six analytes in urine sample. The recoveries at three spiked levels ranged from 77.5 to 97.1%. Besides, the intra column‐to‐column (n = 3) and inter batch‐to‐batch (n = 3) precisions were ≤ 9.8%. The developed method was successfully applied for the determination of hydroxy polycyclic aromatic hydrocarbons in urine samples of coke oven workers.