Column-switching HPLC Method Research Articles

Characterization of insulin-loaded liposome using column-switching HPLC

We evaluated the drug-encapsulation state of insulin (INS)-loaded liposome using a novel column-switching HPLC system that can automatically separate unloaded drug from encapsulated drug by hydrophobic interaction. When the INS-loaded liposome was dispersed in water (pH 7.4), the encapsulation efficiency (EE) obtained by the column-switching HPLC system was consistent with that obtained by a conventional ultracentrifugation method. However, the INS-loaded liposome dispersed in 0.1% acetic acid (pH 3.3) showed disagreement between the EEs obtained by both methods. Considering the results of particle size, zeta potential, and transmission electron microscope (TEM) observations, we hypothesized that the column-switching HPLC method was able to distinguish INS adsorbed onto the liposome surface from the encapsulated INS, although an ultracentrifugation method precipitated the adsorbed INS onto the liposome surface along with the encapsulated INS. Therefore, the novel column-switching HPLC system may be a more accurate and useful technique for characterization and optimization of the INS-loaded liposome formulation.

Pretherapeutic uracil and dihydrouracil levels in saliva of colorectal cancer patients are associated with toxicity during adjuvant 5-fluorouracil-based chemotherapy.

5-fluorouracil (5-FU) competes with uracil (Ura) as a substrate for dihydropyrimidine dehydrogenase (DPD). Low DPD activity impairs breakdown of Ura to dihydrouracil (UH₂) and is associated with toxicity during 5-FU-based chemotherapy. Calculation of the 5-FU dose is based on body surface area, and new tools are needed to individualize treatment. The aim of study was to measure Ura and UH₂ in saliva of patients with colorectal cancer and relate levels to treatment-induced toxicity. Saliva was collected from 73 patients with stage III colorectal cancer prior to adjuvant 5-FU-based treatment. Ura and UH₂ were analyzed by a column-switching HPLC method. Toxicity was evaluated before each treatment cycle and the highest grade was noted at end of treatment. Toxicity was more common and severe among women compared with men. The Ura and UH₂ concentrations in saliva were 5.0 ± 6.8 and 5.0 ± 4.0 nmol/ml, respectively. The UH₂/Ura ratio was lower in women compared with men (1.2 ± 1.0 and 2.2 ± 2.5, respectively, p = 0.0026). Patients who needed to reduce the drug dose during treatment (or terminate treatment) due to toxicity had a lower ratio (1.3 ± 0.85) compared to patients who completed treatment without dose reduction (4.1 ± 4.3, p < 0.0001). Sampling of saliva is a quick, noninvasive, safe and painless process that gives information about patients Ura and UH₂ levels prior to chemotherapeutical treatment. This information may be useful in order to predict and prevent occurrence of treatment-related toxicities which otherwise may limit drug administration.

Rapid Determination of Ginkgolic Acids in Ginkgo biloba Leaf Using Online Column Switching High-Performance Liquid Chromatography-Diode Array Detection and Confirmation by Liquid Chromatography-tandem Mass Spectrometry

In this study, an improved method for the quantitative analysis of ginkgolic acids (GAs) in Ginkgo biloba leaf extract was developed. The samples were extracted with a mixture of chloroform and 50 % ethanol, after which the chloroform extract was dried and reconstituted in methanol. GAs with 13:0, 15:1, and 17:1 in the extract were successfully separated within 40 min and determined with high throughput performance using an online column-switching HPLC method using an SP column C8 SG80 (4.6 × 150 mm, 5 µm) and a Cadenza 5CD C18 column (4.6 × 150 mm, 3 µm). The developed HPLC method was validated for Ginkgo biloba leaf extract. The validation parameters were specificity, linearity, precision, accuracy, and limits of detection and quantitation (LODs and LOQs, respectively). It was found that all of the calibration curves showed good linearity (r 2 > 0.9993) within the tested ranges. The LODs and LOQs were all lower than 0.04 µg/mL. The established method was found to be simple, rapid, and high throughput for the quantitative analysis of GAs in ten commercial Ginkgo biloba leaf extract and dietary supplements. The samples were also analyzed in LC-electrospray ionization (ESI) tandem mass spectrometry (MS/MS) - multiple-ion reaction monitoring (MRM) mode to confirm the identification results that were obtained by the column switching HPLC-DAD method. The developed method is considered to be suitable for the routine quality control and safety assurance of Ginkgo biloba leaf extract.

Preparation of clenbuterol imprinted monolithic polymer with hydrophilic outer layers by reversible addition–fragmentation chain transfer radical polymerization and its application in the clenbuterol determination from human serum by on-line solid-phase extraction/HPLC analysis

A novel imprinted monolithic material with the ability of protein exclusion was developed for the selective extraction of clenbuterol (CLE) from biological samples by direct injection in the HPLC analysis. The material has an imprinted inner structure and hydrophilic outer layer. The reversible addition-fragmentation chain transfer (RAFT) polymerization was employed in the material preparation by a two-step procedure. In the first step, clenbuterol imprinted monolithic polymer was synthesized by combining the molecular imprinting and the RAFT polymerization techniques. The resulting monolithic polymer has a RAFT chain transfer agent (trithioester groups) in its structure, which was used to graft poly(glycerol mono-methacrylate) [pGMMA] in the second step by post-RAFT polymerization. The hydrophilic pGMMA layers grafted on the surface of the imprinted monolith created barriers for protein diffusion. More than 90% of bovine serum albumin can be excluded from the pGMMA coated monolithic column. Meanwhile the clenbuterol was retained selectively with a large retention factor. The result indicated that the column, denoted as RA-MIM, has both the merits of a molecularly imprinted polymer and restricted access material. By using RA-MIM as the solid-phase extraction pre-column, an on-line column-switching HPLC method for the determination of clenbuterol in human serum has been established and validated. The recoveries of clenbuterol from the serum were 87.3-96.9% in the spiked level 2-1000 ng mL(-1). Both good linearity (R = 0.999) and acceptable reproducibility (RSD < 7.0%) were obtained. The limit of detection and the limit of quantitation were 0.7 ng mL(-1) and 2.0 ng mL(-1) respectively, which is sensitive in terms of UV detection. The results have demonstrated that the RAFT polymerization can be used to synthesize bi-functional monolithic columns by using its living reaction property. The resulting RA-MIM in this research can be used for efficient clenbuterol determination by HPLC from biological samples.

Bioequivalence of Hana Loxoprofen Sodium Tablet to Dongwha Loxonin®Tablet (Loxoprofen Sodium 60 mg)

Loxoprofen sodium, a 2-phenylpropionate non-steroidal anti-inflammatory drug (NSAID), has marked analgesic and antipyretic activities and relatively weak gastrointestinal ulcerogenicity. The purpose of the present study was to evaluate the bioequivalence of two loxoprofen sodium tablets, Hana loxoprofen sodium tablet (Hana Pharm. Co., Ltd.) and Dongwha Loxonin(R) tablet (Dongwha Pharm. Co., Ltd.), according to the guidelines of the Korea Food and Drug Administration (KFDA). The in vitro release of loxoprofen from the two loxoprofen sodium formulations was tested using KP IX Apparatus II method with various dissolution media. Twenty four healthy Korean male volunteers, 22.83±1.86 years in age and 69.92±9.14 kg in body weight, were divided into two groups and a randomized 2×2 crossover study was employed. After a single tablet containing 60 mg as loxoprofen sodium was orally administered, blood samples were taken at predetermined time intervals and the concentrations of loxoprofen in serum were determined using a online column-switching HPLC method with UV/Vis detection. The dissolution profiles of two formulations were similar in all tested dissolution media. The pharmacokinetic parameters such as AUCt, Cmax and Tmax were calculated, and computer programs (Equiv Test and K-BE Test 2002) were utilized for the statistical analysis of the parameters using logarithmically transformed AUCt, Cmax and un-transformed Tmax. The results showed that the differences between two formulations based on the reference drug, Dongwha Loxonin(R) tablet, were 2.03, 2.99 and -9.49% for AUCt, Cmax, and Tmax, respectively. There were no sequence effects between two formulations in these parameters. The 90% confidence intervals using logarithmically transformed data were within the acceptance range of log0.8 to log1.25 (e.g., log0.9831~log1.0535 and log0.9455~log1.1386 for AUCt and Cmax, respectively). Thus, the criteria of the KFDA bioequivalence guideline were satisfied, indicating Hana loxoprofen sodium tablet was bioequivalent to Dongwha Loxonin(R) tablet.

Determination of time-dependent accumulation of d-lactate in the streptozotocin-induced diabetic rat kidney by column-switching HPLC with fluorescence detection

For better understanding the complete metabolism and the physiological role of d-lactate, the concentrations of d-lactate in the serum, liver and kidney of normal and diabetic rats were determined by our established column-switching HPLC method with pre-column fluorescence derivatization. Eight-week-old male Sprague–Dawley rats were administered with streptozotocin (STZ) (80 mg/kg) or citrate buffer intraperitoneally. The tissues were then removed and homogenized after 4, 8, 12 and 16 weeks of drug administration, respectively. The homogenates were centrifuged at 1200 × g for 10 min, then the supernatants were derivatized with a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), separated on an ODS column followed by a Chiralpak AD-RH chiral column for enantioseparation. The results showed that the d-lactate content elevated in all the 3 examined tissues under diabetic stages. In addition, d-lactate concentrations in rat kidney were accumulated significantly and time-dependently in diabetic groups after receiving STZ for 4, 8, 12 and 16 weeks (2.99, 13.11, 18.19, 23.23 vs. 0.79 μmol/mg protein as control group). Moreover, the kidney of induced 12-week diabetic rat renal showed some histological changes of progressive diabetic nephropathy. The results suggest that d-lactate may be used as a marker of diabetic nephropathy.

A sensitive column‐switching HPLC method for aripiprazole and dehydroaripiprazole and its application to human pharmacokinetic studies

A simple and sensitive column-switching HPLC-UV method was developed for the simultaneous determination of aripiprazole, a novel atypical antipsychotic drug, and its active metabolite, dehydroaripiprazole in human plasma. Aripiprazole, its active metabolite and 7-[5-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]pentyloxy]-3,4-dihydro-2(1H)-quinolinone (OPC-14558) as an internal standard were extracted from 1 mL of plasma using a mixture of chloroform/n-heptane (3:7, v/v), and the extract was injected into a column I (TSK BSA-ODS/S precolumn, 5 μm) for cleanup and column II (C(18) STR ODS-II analytical column, 5 μm) for separation. Peaks were detected with an UV detector set at a wavelength of 254 nm, and the total time for chromatographic separation was ∼20 min. Mean absolute recoveries were 74.0 and 74.7% for aripiprazole and dehydroaripiprazole, respectively. Intra- and inter-day CVs were less than 7.5 and 7.1% for aripiprazole concentrations ranging from 2 to 600 ng/mL, and 9.2 and 4.5% for dehydroaripiprazole concentrations ranging from 2 to 160 ng/mL. The validated concentration ranges for this method were 1-500 ng/mL and the limits of detection were 0.5 ng/mL for both aripiprazole and dehydroaripiprazole. This method was applied to pharmacokinetic study in human volunteers and patients taking aripiprazole.

Simple determination of azasetron in rat plasma by column‐switching high‐performance liquid chromatography

For the quantification of azasetron in rat plasma samples, a column-switching HPLC method was developed and validated. Following dilution of plasma samples with mobile phase A (17 mM potassium phosphate buffer (pH 3.0)) and simple protein precipitation by addition of perchloric acid (60%), the mixture was directly injected onto the pre-column. After endogenous plasma substances were eluted to waste, the analyte was transferred to the trap column by switching the system. Then, the analyte was back-flushed to the analytical column for separation with mobile phase B (a 22:78 v/v mixture of acetonitrile and 17 mM potassium phosphate buffer (pH 3.0)) and detected at 250 nm using a photodiode array detector. A linear standard curve was obtained in the concentration range of 10-800 ng/mL with the correlation coefficient (r) of 0.9998. The intra- and inter-day precision and accuracy values for azasetron were in the ranges of 0.3-12.9% and 89.7-101.4%, respectively. The method was valid in terms of specificity, precision, and accuracy. In addition, this efficient analytical method was successfully applied to determine plasma concentrations of azasetron following oral administration of azasetron at a dose of 4.0 mg/kg to rats.

Determination of sulfonamides in bovine milk with column-switching high performance liquid chromatography using surface imprinted silica with hydrophilic external layer as restricted access and selective extraction material

A novel restricted access-molecularly imprinted material (RA-MIP) with selectivity for sulfonamides was synthesized using initiator-transfer agent-terminator (iniferter) method, a “living”/controlled radical polymerization technique. The material was prepared by grafting two layers with different functions on the silica support. To perform a “grafting from” polymerization, iniferter was immobilized on the surface of silica. The internal sulfamethazine imprinted polymer and the external poly(glycidyl methacrylate) [poly(GMA)] were then grafted successively. The hydrophilic structures were formed on the external layer of the material by the hydrolysis of the linear poly(GMA) for protein removal. The result has shown that this restricted access-MIP grafted silica (RA-MIP-SG) not only has the selectivity for the template and its analog, but also has the ability of exclusion for bovine serum albumin (BSA). It indicated that the material possesses both properties of molecularly imprinted polymer (MIP) and restricted access material (RAM). Using RA-MIP-SG as pre-column, a column-switching HPLC method was established for the determination of sulfonamides in bovine milk. Direct sample injection was performed in the analysis, which provides a convenient analytical procedure. Good linearity in the range of 2–400 ng mL −1 ( R > 0.998) has been obtained for seven sulfonamides in the study. The recoveries of all the analytes at three concentration levels are between 93% and 107% with the RSD < 8.0%. The limits of quantification and limits of detection are less than or equal to 2.7 ng mL −1 and 0.8 ng mL −1 respectively. It demonstrated this RA-MIP-SG can be applied in sample extraction and clean-up for the determination of sulfonamides in bovine milk by direct injection and column-switching HPLC with good efficiency and reliability.

Determination of neotame in beverages, cakes and preserved fruits by column-switching high-performance liquid chromatography

A column-switching HPLC method for the determination of neotame in beverages, cakes and preserved fruits was developed. After pre-treatment using a Waters Oasis HLB cartridge, the sample solution was separated on two C18 columns using a column-switching technique with a six-port valve. UV detection was performed at 210 nm. The effects of eluent composition and eluate volume on the retention and elution of neotame on SPE cartridge were investigated. The method is simple, rapid, sensitive and has good reproducibility. RSD was lower than 5% (n = 5). The calibration curve of neotame was in the range 5–100 µg/ml with good linearity (r 2 = 0.999). Because neotame was concentrated 10 times from an original sample to a sample solution by solid-phase extraction (SPE), the limit of quantification (LOQ) of the method was 0.5 mg/kg. The recovery yields of neotame spiked in foods was >92% with a coefficient of variation <3.2%. The proposed column-switching HPLC method can be successfully used to determine neotame in routine inspection work.

Decomposition of 3′‐azido‐2′,3′‐dideoxythymidine 5′‐monophosphate (AZTMP) prodrugs in biological media studied by on‐line solid‐phase extraction coupled to liquid chromatography mass spectrometry

Using a column-switching HPLC method previously described, we studied the behavior of some mononucleotide prodrugs (pronucleotides) of 3'-azido-2',3'-dideoxythymidine in various biological media. From UV data, this method allowed quantification of transient metabolites resulting from prodrug bioconversion. The kinetic data related to the successive steps were calculated according to pseudo-first-order kinetic models and optimized using mono- or poly-exponential regressions. Various metabolites were identified by co-injection with authentic samples and/or ESI-MS coupling. The results led us to propose, for each considered pronucleotide, a global decomposition pathway ending in the selective delivery of the corresponding mononucleotide. Associated to the determination of other parameters (lipophilicity, aqueous solubility), the present study contributes to the search of suitable pharmacological properties for further in vivo evaluations.

Determination of the active metabolites of sibutramine in rat serum using column‐switching HPLC

A simple and direct analysis using column-switching HPLC method was developed and validated for the quantification of active metabolites of sibutramine, N-mono-desmethyl metabolite (metabolite 1, M1) and N-di-desmethyl metabolite (metabolite 2, M2) in the serum of rats administered sibutramine HCl (5.0 mg/kg, p.o.). Rat serum was directly injected onto the precolumn without sample prepreparation step following dilution with mobile phase A, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (8.3:4.5:87.2 by volume). After the endogenous serum components were eluted to waste, the system was switched and the analytes were eluted to the trap column. Active metabolites M1 and M2 were then back-flushed to the analytical column for separation with mobile phase B, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (35.8:19.2:45 by volume) and detected at 223 nm. The calibration curves of active metabolites M1 and M2 were linear in the range of 0.1-1.0 microg/mL and 0.15-1.8 microg/mL. This method was fully validated and shown to be specific, accurate (10.4-10.7% error), and precise (1.97-8.79% CV). This simple and rapid analytical method using column-switching appears to be useful for the pharmacokinetic study of active metabolites (M1 and M2) of sibutramine.

Improved HPLC column‐switching determination of coenzyme Q and Vitamin E in plasma

A novel isocratic modified column-switching HPLC method for automated quantitative analysis of Vitamin E and Coenzyme Q, in the reduced and oxidized form, is described. Many column-switching HPLC methods are found in the literature, also for determining antioxidant substances, but we developed a different system of column-switching. An empty column, 5 cm long, was connected to the switching valve, before the sample loop and the extraction column. The sample loop was connected directly after the empty column. The inserted column, containing about 1.4 ml of the extraction eluent simulated a gradient elution, enhancing sensitivity and resolution. When switching the columns, the empty column is placed right before the extraction column and acts as a static mixer for the extraction phase and the incoming analytical phase. Samples were cleaned from interfering compounds by transfer onto a extraction-column, using a C-8 silica. Separation was performed onto an analytical column C18 3 m icrom, 150 mm x 4.6 mm at a flow rate of 1.0 ml/min with 20 mmol/l lithium perchlorate/perchloric acid, pH3.0 in Ethanol as analytical eluent. Detection was performed with a ESA Coulochem 5100 A model. The method was found to be suitable for automated analysis of Coenzyme Q, reduced and oxidized form, and Vitamin E in serum.

Rapid, simple and simultaneous measurement of kynurenine and tryptophan in plasma by column switching-HPLC method

Abstract We established a highly reproducible, simple and rapid simultaneous measurement method in which blood plasma can be directly injected into high-performance liquid chromatography (HPLC) without conducting conventional extraction of kynurenine (Kyn) and tryptophan (Trp) from blood plasma. In this method, Kyn and Trp were first separated from proteins present in blood plasma using a pre-column. Then, by column-switching, the separated Kyn and Trp were automatically injected into a separation column. The recoveries of Kyn and Trp determined in blood plasma by this method were 100.0 ± 0.9 and 100.0 ± 1.4%, respectively.

Simultaneous Determination of Angiotensin I-Converting Enzyme Inhibitory Peptides in Tryptic Casein Hydrolysate by High-Performance Liquid Chromatography Combined with a Replicate Heart-Cut Column-Switching Technique

A replicate heart-cut column-switching HPLC method combined with two switching valves was newly developed for the simultaneous determination of three antihypertensive peptides (Ala-Phe, Tyr-Pro, and Trp-Tyr) in tryptic casein hydrolysate in one run-in assay. After a first separation on an octadecyl silane (ODS) column, heart-cuts of each peptide were individually separated on a subsequent analytical ODS column: 26% acetonitrile for Ala-Phe and Tyr-Pro (32% for Trp-Tyr) in 0.1% trifluoroacetic acid containing 10 mM sodium 1-octanesulfonate at 0.8 mL/min. Ala-Phe, Tyr-Pro, and Trp-Tyr in casein hydrolysate were determined within 70 min to be 0.377 +/- 0.037 mg/g, 2.50 +/- 0.26 mg/g, and 0.096 +/- 0.008 mg/g, respectively.

Determination of Antihypertensive Small Peptides, Val-Tyr and Ile-Val-Tyr, by Fluorometric High-Performance Liquid Chromatography Combined with a Double Heart-Cut Column-Switching Technique

A double column-switching HPLC method with naphthalene-2,3-dialdehyde (NDA) was applied for determination of two plasma antihypertensive peptides, Val-Tyr (VY) and Ile-Val-Tyr (IVY). After a first separation on a Phe-ODS column, double heart-cuts of the retention time corresponding to NDA-VY and NDA-IVY elutions were successfully separated on an analytical ODS column: 60% acetonitrile in 0.1% trifluoroacetic acid containing 5 mM sodium 1-octanesulfonate at 1.0 mL/min. Within-run coefficients of variation were 1.73% and 4.73% for VY and IVY, respectively.

Intravenous administration of 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (gamma-CEHC) to rats and determination of its plasma concentration and urinary sodium excretion.

A natriuretic hormone, 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (gamma-CEHC) was administered intravenously to male Sprague-Dawley rats and the plasma concentration of gamma-CEHC along with urinary sodium (Na+) excretion was investigated. The plasma gamma-CEHC concentrations were fluorimetrically determined by a column-switching HPLC method consisting of both phenyl and octadecyl silica columns, following a pre-column fluorescence derivatization with a fluorescence reagent, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ). In rats fed with a high-NaCl (8.0%) diet, plasma gamma-CEHC concentrations rapidly decreased by 20% in 15-45 min after the administration of gamma-CEHC, while Na+ excretion gradually increased with time. Considering these results, the Na+ excretion effect appeared not to be associated with plasma gamma-CEHC concentration. In addition, attempts were made to examine a main urinary metabolite of gamma-CEHC, a large amount of 6-O-sulfated gamma-CEHC found to be present in the urine using an HPLC-tandem mass spectrometry. Thus, it is plausible that gamma-CEHC was easily metabolized to 6-O-sulfated metabolite and excreted into urine in rats.

스프렌딜 지속정(펠로디핀 5 mg)에 대한 스타핀 지속정의 생물학적동등성

Felodipine is a calcium antagonist that lowers blood pressure by reducing peripheral resistance by meas of a direct, selective action on smooth muscle in arterial resistance vessels. Futhermore, it have been approved for the effective in angina pectoris and cardiac failure. The purpose of the present study was to evaluate the bioequivalence of two felodipine extended release (ER) tablets, Splendil (YuHan Corporation) and Stapin (Hana Pharmaceutial Co., Ltd.), according to the guidelines of Korea Food and Drug Administration (KFDA). The felodipine release from the two felodipine formulations in vitro was tested using KP VIII Apparatus II method at pH 6.5 buffer solution. Twenty six healthy male subjects, years in age and in body weight, were divided into two groups and a radomized cross-over study was employed. After two tablets containing 5 mg as felodipine were orally administered, blood sample was taken at predetermined time intervals and the concentrations of felodipine in serum were determined using column-switching HPLC method with UV detector. The dissolution profiles of two formulations were similar at pH 6.5 buffer solution. Besides, the pharmacokinetic parameters such as were calculated and ANOVA test was utilized for the statistical analysis of the parameters using logarithmically transformed and untransformed . The results showed that the differences between two formulations based on the Splendil were 2.53%, 1.32% and 18.32% for , respectively. There were no sequence effects between two formulations in these parameters. The 90% confidence intervals using logarithmically transformed data were within the acceptance rage of log(0.86) to log(1.25) . Thus, the criteria of the KFDA guidelines for the bioequivalence was satisfied, indicating Stapin ER tablet and Splendil ER tablet are bioequivalent.

Liquid chromatography determination of 10-hydroxycamptothecin in human serum by a column-switching system containing a pre-column with restricted access media and its application to a clinical pharmacokinetic study

A simple, rapid, sensitive column-switching HPLC method is described for the analysis of the 10-hydroxycamptothecin (HCPT) in human serum. A pre-column containing restricted access media (RAM) is used for the sample clean-up and trace enrichment and is combined with a C 18 column for the final separation. The analytical time is 8 min. The HCPT is monitored with fluorescence detector, excitation and emission wavelengths being 385 and 539 nm, respectively. There is a linear response range of 1–1000 ng/ml with correlation coefficient of 0.998 while the limit of quantification is 0.1 ng/ml. The intra-day and inter-day variations are less than 5%. This analytic procedure has been applied to a pharmacokinetic study of HCPT in clinical patients and the pharmacokinetic parameters of one-compartment model are calculated.

Measurement of bisphenol A levels in human blood serum and ascitic fluid by HPLC using a fluorescent labeling reagent

A sensitive column-switching HPLC method with fluorescence detection was developed for the determination of bisphenol A (BPA) in human blood serum and ascitic fluid samples. 4-(4,5-Diphenyl-1 H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was used as the fluorescent label, and the excess reagent was removed by a column-switching technique. Liquid–liquid extraction with chloroform was used for the pretreatment of serum and ascitic fluid samples. BPA in both the samples could be determined in the concentration range of 0.1–7.0 ppb with the detection limit of 0.04 ppb at a signal-to-noise ratio of 3. The recoveries of BPA spiked to serum and ascitic fluid were 78.6 and 77.7%, respectively. The mean concentrations of BPA ( n=9) in maternal and umbilical cord blood sera obtained from healthy pregnant women were 0.46±0.20 and 0.62±0.13 ppb, respectively. BPA levels ( n=21) in blood sera and ascitic fluid obtained from the patients with sterility were also determined to be 0.46±0.20 and 0.56±0.19 ppb, respectively. Relationships of BPA concentrations were observed between maternal and umbilical cord blood serum samples ( r=0.626), as well as blood serum and ascitic fluid samples ( r=0.785).