İDRARDA 3-FENİLPİRÜVİK ASİT TAYİNİ İÇİN SIVI-SIVI EKSTRAKSİYONUNA VE LC-MS/MS'E DAYALI BİR ANALİTİK YÖNTEMİN GELİŞTİRİLMESİ
Abstract
Amaç : Bu çalışmanın amacı Fenilalanin Hidroksilaz Enzimi eksikliği sonucunda ortaya çıkan metabolik bir hastalık olan fenilketonürinin tanısında önemli bir yeri olan 3-fenilpirüvik asit tayini için hızlı, kesin, spesifik bir LC MS MS yöntemi geliştirilmesidir.
Gereç ve Yöntem : Analitik ölçümler Acqutiy UPLC MS MS (Waters Xevo TQD) cihazı ile yapılmıştır. Kromatografik ayrım Acquity UPLC Phenyl (50 mm × 2.1 mm, 1.7 μm) kolonu ile metanol ve % 0,1 formik asit içeren suyun mobil faz olarak kullanıldığı gradient elüsyon programı ile yapılmıştır. Proje kapsamında ilk olarak örnek hazırlama adımlarına odaklanılmış olup dispersiv sıvı sıvı ekstraksiyon ve geleneksel sıvı ekstraksiyon yöntemi denenmiştir. En iyi sonuçlar diklorometanın ekstraksiyon çözgeni olarak kullanıldığı geleneksel sıvı sıvı ekstraksiyon yönteminde elde edilmiştir. Ayrıca bu çalışmadan 3-Fenilpirüvik asit tayini için trans sinamik asit internal standart olarak test edilmiş ve validasyon adımlarından geçmiştir.
Bulgular : Geliştirilen LC MS MS yönteminin doğrusal aralığı 0.009-5 M arası olarak bulunurken tayin limit ise 0.001 M olarak bulunmuştur. Gün içi tekrarlanabilirlik 3 seviye için %7’nin altında, günler arası tekrarlanabilirlik ise %10’un altında bulunmuştur. Yöntemin doğruluğunu gösteren geri elde denemesinde ise gene 3 seviye için idrar örneğinde sonuçlar %97-%103 aralığında, örnek hazırlama aşamaları sonucu elde edilen son matriksin ne kadar temiz olduğunu gösteren matriks etkisi denemesinde kaydadeğer bir iyon baskılama olmadığı saptanmıştır.
References
- Qiu F, Yang C, Yuan L, Xiang D, Lan X, Chen M, et al. A Phenylpyruvic Acid Reductase Is Required for Biosynthesis of Tropane Alkaloids. Org Lett. 2018 Dec 21;20(24):7807–10.
- Cleary MA. Phenylketonuria. Paediatr Child Health. 2011 Feb 1;21(2):61–4.
- Hanley WB. Adult phenylketonuria. Am J Med. 2004 Oct 15;117(8):590–5.
- Blau N, Spronsen FJ van, Levy HL. Phenylketonuria. The Lancet. 2010 Oct 23;376(9750):1417–27.
- MacDonald A, Rocha JC, van Rijn M, Feillet F. Nutrition in phenylketonuria. Mol Genet Metab. 2011 Jan 1;104:S10–8.
- Hafid NA, Christodoulou J. Phenylketonuria: a review of current and future treatments. Transl Pediatr. 2015 Oct;4(4):30417–30317.
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- Nakahara T, Ishida J, Yamaguchi M, Nakamura M. Determination of α-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence detection. Anal Biochem. 1990 Nov 1;190(2):309–13.
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- Rykowska I, Ziemblińska J, Nowak I. Modern approaches in dispersive liquid-liquid microextraction (DLLME) based on ionic liquids: A review. J Mol Liq. 2018 Jun 1;259:319–39.
Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS/MS
Abstract
Aim: The aim of this study is to develop a rapid, precise, specific LC MS MS method for the determination of 3-phenylpyruvic acid, which has an important place in the diagnosis of phenylketonuria, a metabolic disease resulting from Phenylalanine Hydroxylase Enzyme deficiency.
Materials and Methods : Analytical measurements were made with Acqutiy UPLC MS MS (Waters Xevo TQD). The chromatographic separation was operated on an Acquity UPLC Phenyl column (50 mm × 2.1 mm, 1.7 μm) with gradient elutionusing 0.1% formic acid containing water and methanol as the mobile phase. Within the scope of the study, firstly, sample preparation steps were focused and dispersive liquid liquid extraction and traditional liquid extraction methods were examined. The best results were obtained in the conventional liquid liquid extraction method, in which dichloromethane was used as the extraction solvent. In addition, in this study, trans cinnamic acid was tested as an internal standard for the determination of 3-phenylpyruvic acid and passed the validation steps.
Results : The linear range of the developed LC MS MS method was found to be between 0.009-5 M, while the detection limit was found to be 0.001 M. Intraday repeatability was below 7% for 3 levels, and interday repeatability was below 10%. In the recovery trial, which showed the accuracy of the method, the results in the urine sample for 3 levels were in the range of 97%-103%, and there was no significant ion suppression in the matrix effect trial, which showed how clean the final matrix obtained as a result of the sample preparation steps was.
Conclusion: As a result, a fast, inexpensive LC MS MS method has been developed for 3-Phenylpyruvic acid.
References
- Qiu F, Yang C, Yuan L, Xiang D, Lan X, Chen M, et al. A Phenylpyruvic Acid Reductase Is Required for Biosynthesis of Tropane Alkaloids. Org Lett. 2018 Dec 21;20(24):7807–10.
- Cleary MA. Phenylketonuria. Paediatr Child Health. 2011 Feb 1;21(2):61–4.
- Hanley WB. Adult phenylketonuria. Am J Med. 2004 Oct 15;117(8):590–5.
- Blau N, Spronsen FJ van, Levy HL. Phenylketonuria. The Lancet. 2010 Oct 23;376(9750):1417–27.
- MacDonald A, Rocha JC, van Rijn M, Feillet F. Nutrition in phenylketonuria. Mol Genet Metab. 2011 Jan 1;104:S10–8.
- Hafid NA, Christodoulou J. Phenylketonuria: a review of current and future treatments. Transl Pediatr. 2015 Oct;4(4):30417–30317.
- Brown CS, Lichter-Konecki U. Phenylketonuria (PKU): A problem solved? Mol Genet Metab Rep. 2016 Mar 1;6:8–12.
- Pitt JJ. Principles and Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry. Clin Biochem Rev. 2009 Feb;30(1):19–34.
- Whitmire M, Ammerman J, Lisio PD. LC-MS/MS Bioanalysis Method Development, Validation, and Sample Analysis: Points to Consider When Conducting Nonclinical and Clinical Studies in Accordance with Current Regulatory Guidances. J Anal Bioanal Tech [Internet]. 2011 [cited 2024 Feb 23];01(01). Available from: https://www.omicsonline.org/lc-ms-ms-bioanalysis-method-development-validation-and-sample-analysispoints-to-consider-2155-9872.S4-001.php?aid=1745
- Kushnir MM, Rockwood AL, Roberts WL, Yue B, Bergquist J, Meikle AW. Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories. Clin Biochem. 2011 Jan 1;44(1):77–88.
- Wieling J. LC-MS-MS experiences with internal standards. Chromatographia. 2002 Jan 1;55(1):S107–13.
- Human Metabolome Database: Showing metabocard for trans-Cinnamic acid (HMDB0000930) [Internet]. [cited 2024 Feb 23]. Available from: https://hmdb.ca/metabolites/HMDB0000930
- Basaria S, Wahlstrom JT, Dobs AS. Clinical review 138: Anabolic-androgenic steroid therapy in the treatment of chronic diseases. J Clin Endocrinol Metab. 2001 Nov;86(11):5108–17.
- Human Metabolome Database: Showing metabocard for Phenylpyruvic acid (HMDB0000205) [Internet]. [cited 2024 Feb 23]. Available from: https://hmdb.ca/metabolites/HMDB0000205
- Cassidei L, Dell’atti A, Sciacovelli O. Improvement of the FeCl3 test for phenylpyruvic acid. Clin Chim Acta Int J Clin Chem. 1978 Dec 1;90(2):121–7.
- Sano A, Ogawa M, Takitani S. Fluorometric determination of phenylpyruvic acid with 1,4-dimethyl-3- carbamoylpyridinium chloride. Chem Pharm Bull (Tokyo). 1987 Sep;35(9):3746–9.
- Hirata T, Kai M, Kohashi K, Ohkura Y. Determination of phenylpyruvic acid in urine and serum by highperformance liquid chromatography with fluorescence detection. J Chromatogr. 1981 Nov 13;226(1):25–31.
- Nakahara T, Ishida J, Yamaguchi M, Nakamura M. Determination of α-keto acids including phenylpyruvic acid in human plasma by high-performance liquid chromatography with chemiluminescence detection. Anal Biochem. 1990 Nov 1;190(2):309–13.
- Hara S, Takemori Y, Yamaguchi M, Nakamura M, Ohkura Y. Determination of alpha-keto acids in serum and urine by high-performance liquid chromatography with fluorescence detection. J Chromatogr. 1985 Nov 8;344:33–9.
- Noguchi K, Mizukoshi T, Miyano H, Yamada N. Development of a New LC-MS/MS Method for the Quantification of Keto Acids. Chromatography. 2014;35(3):117–23.
- Hsu LW, Lin YH, Guo JY, Chen CF, Chou YJ, Yeh YC. Simultaneous Determination of l-Phenylalanine, Phenylethylamine, and Phenylacetic Acid Using Three-Color Whole-Cell Biosensors within a Microchannel Device. ACS Appl Bio Mater. 2020 Aug 17;3(8):5120–5.
- Bioanalytical method validation - Scientific guideline | European Medicines Agency [Internet]. [cited 2024 Feb 23]. Available from: https://www.ema.europa.eu/en/bioanalytical-method-validation-scientific-guideline
- Miller JN, Miller JC. Statistics and Chemometrics for Analytical Chemistry. Pearson/Prentice Hall; 2005. 296 p.
- ICH Q2(R2) Validation of analytical procedures - Scientific guideline | European Medicines Agency [Internet]. [cited 2024 Feb 23]. Available from: https://www.ema.europa.eu/en/ich-q2r2-validation-analytical-proceduresscientific-guideline
- Rezaee M, Assadi Y, Milani Hosseini MR, Aghaee E, Ahmadi F, Berijani S. Determination of organic compounds in water using dispersive liquid–liquid microextraction. J Chromatogr A. 2006 May 26;1116(1):1– 9.
- Rykowska I, Ziemblińska J, Nowak I. Modern approaches in dispersive liquid-liquid microextraction (DLLME) based on ionic liquids: A review. J Mol Liq. 2018 Jun 1;259:319–39.
Details
| Primary Language | English |
|---|---|
| Subjects | Health Care Administration |
| Journal Section | Research Articles |
| Authors | |
| Publication Date | March 19, 2024 |
| Submission Date | February 15, 2023 |
| Published in Issue | Year 2024 |