Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples
A simple reversed-phase HPLC method for measuring hepatic levels of acetaminophen- (APAP-) protein adduct following an overdose of APAP was developed. An aliquot of liver homogenate in phosphate-buffered saline pH 7.4 (PBS) was placed on a Nanosep centrifugal device, which was centrifuged to obtain...
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Wiley
2012-01-01
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| Series: | The Scientific World Journal |
| Online Access: | http://dx.doi.org/10.1100/2012/145651 |
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| author | Miteshkumar Acharya Cesar A. Lau-Cam |
| author_facet | Miteshkumar Acharya Cesar A. Lau-Cam |
| author_sort | Miteshkumar Acharya |
| collection | DOAJ |
| description | A simple reversed-phase HPLC method for measuring hepatic levels of acetaminophen- (APAP-) protein adduct following an overdose of APAP was developed. An aliquot of liver homogenate in phosphate-buffered saline pH 7.4 (PBS) was placed on a Nanosep centrifugal device, which was centrifuged to obtain a protein residue. This residue was incubated with a solution of p-aminobenzoic acid (PABA), the internal standard, and bacterial protease in PBS, transferred to a Nanosep centrifugal device, and centrifuged. A 100 μL portion of the filtrate was analyzed on a YMC-Pack ODS-AMQ C18 column, using 100 mM potassium dihydrogen phosphate-methanol-acetic acid (100 : 0.6 : 0.1) as the mobile phase, a flow rate of 1 mL/min, and photometric detection at 254 nm. PABA and APAP-cystein-S-yl (APAP-Cys) eluted at ~14.7 min and 22.7 min, respectively. Method linearity, based on on-column concentrations of APAP-Cys, was observed over the range 0.078–40 μg. Recoveries of APAP-Cys from spiked blank liver homogenates ranged from ~83% to 91%. Limits of detection and of quantification of APAP-Cys, based on column concentrations, were 0.06 μg and 0.14 μg, respectively. RSD values for interday and intraday analyses of a blank liver homogenate spiked with APAP-Cyst at three levels were, in all cases, ≤1.0% and <1.5%, respectively. The proposed method was found appropriate for comparing the antidotal properties of N-acetylcysteine and taurine in a rat model of APAP poisoning. |
| format | Article |
| id | doaj-art-8194d4fc2eee49a29dd7b2e0311ec954 |
| institution | OA Journals |
| issn | 1537-744X |
| language | English |
| publishDate | 2012-01-01 |
| publisher | Wiley |
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| series | The Scientific World Journal |
| spelling | doaj-art-8194d4fc2eee49a29dd7b2e0311ec9542025-08-20T02:08:27ZengWileyThe Scientific World Journal1537-744X2012-01-01201210.1100/2012/145651145651Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver SamplesMiteshkumar Acharya0Cesar A. Lau-Cam1Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, Jamaica, NY 11439, USADepartment of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, Jamaica, NY 11439, USAA simple reversed-phase HPLC method for measuring hepatic levels of acetaminophen- (APAP-) protein adduct following an overdose of APAP was developed. An aliquot of liver homogenate in phosphate-buffered saline pH 7.4 (PBS) was placed on a Nanosep centrifugal device, which was centrifuged to obtain a protein residue. This residue was incubated with a solution of p-aminobenzoic acid (PABA), the internal standard, and bacterial protease in PBS, transferred to a Nanosep centrifugal device, and centrifuged. A 100 μL portion of the filtrate was analyzed on a YMC-Pack ODS-AMQ C18 column, using 100 mM potassium dihydrogen phosphate-methanol-acetic acid (100 : 0.6 : 0.1) as the mobile phase, a flow rate of 1 mL/min, and photometric detection at 254 nm. PABA and APAP-cystein-S-yl (APAP-Cys) eluted at ~14.7 min and 22.7 min, respectively. Method linearity, based on on-column concentrations of APAP-Cys, was observed over the range 0.078–40 μg. Recoveries of APAP-Cys from spiked blank liver homogenates ranged from ~83% to 91%. Limits of detection and of quantification of APAP-Cys, based on column concentrations, were 0.06 μg and 0.14 μg, respectively. RSD values for interday and intraday analyses of a blank liver homogenate spiked with APAP-Cyst at three levels were, in all cases, ≤1.0% and <1.5%, respectively. The proposed method was found appropriate for comparing the antidotal properties of N-acetylcysteine and taurine in a rat model of APAP poisoning.http://dx.doi.org/10.1100/2012/145651 |
| spellingShingle | Miteshkumar Acharya Cesar A. Lau-Cam Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples The Scientific World Journal |
| title | Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples |
| title_full | Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples |
| title_fullStr | Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples |
| title_full_unstemmed | Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples |
| title_short | Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples |
| title_sort | simple reversed phase hplc method with spectrophotometric detection for measuring acetaminophen protein adducts in rat liver samples |
| url | http://dx.doi.org/10.1100/2012/145651 |
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