Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS/MS

Erhan Canbay; Zeynep Çelik Canbay; Berrak Yeşilyurt; Serçin Doğan; İrem Arabacı; Ebru Demirel Sezer; Eser Y. Sözmen

doi:10.19161/etd.1250567

TR EN

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.

Keywords

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.

Keywords

References

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Details

Primary Language

English

Subjects

Health Care Administration

Journal Section

Research Article

Authors

Erhan Canbay ^*
0000-0003-0948-1675
Türkiye

Zeynep Çelik Canbay
0000-0001-7159-1088
Türkiye

Berrak Yeşilyurt
0000-0003-4212-0521
Türkiye

Serçin Doğan
0000-0002-9735-8142
Türkiye

İrem Arabacı
0000-0002-9366-701X
Türkiye

Ebru Demirel Sezer
0000-0002-6900-3539
Türkiye

Eser Y. Sözmen
0000-0002-6383-6724
Türkiye

Publication Date

March 19, 2024

Submission Date

February 15, 2023

Acceptance Date

April 19, 2023

Published in Issue

Year 2024 Volume: 63 Number: 1

DOI

https://doi.org/10.19161/etd.1250567

IZ

https://izlik.org/JA89KR57MU

Cite

RIS / Bibtex

APA

Canbay, E., Çelik Canbay, Z., Yeşilyurt, B., Doğan, S., Arabacı, İ., Demirel Sezer, E., & Y. Sözmen, E. (2024). Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS/MS. Ege Tıp Dergisi, 63(1), 1-12. https://doi.org/10.19161/etd.1250567

AMA

1.Canbay E, Çelik Canbay Z, Yeşilyurt B, et al. Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS/MS. EJM. 2024;63(1):1-12. doi:10.19161/etd.1250567

Chicago

Canbay, Erhan, Zeynep Çelik Canbay, Berrak Yeşilyurt, et al. 2024. “Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS MS”. Ege Tıp Dergisi 63 (1): 1-12. https://doi.org/10.19161/etd.1250567.

EndNote

Canbay E, Çelik Canbay Z, Yeşilyurt B, Doğan S, Arabacı İ, Demirel Sezer E, Y. Sözmen E (March 1, 2024) Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS/MS. Ege Tıp Dergisi 63 1 1–12.

IEEE

[1]E. Canbay et al., “Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS/MS”, EJM, vol. 63, no. 1, pp. 1–12, Mar. 2024, doi: 10.19161/etd.1250567.

ISNAD

Canbay, Erhan - Çelik Canbay, Zeynep - Yeşilyurt, Berrak - Doğan, Serçin - Arabacı, İrem - Demirel Sezer, Ebru - Y. Sözmen, Eser. “Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS MS”. Ege Tıp Dergisi 63/1 (March 1, 2024): 1-12. https://doi.org/10.19161/etd.1250567.

JAMA

1.Canbay E, Çelik Canbay Z, Yeşilyurt B, Doğan S, Arabacı İ, Demirel Sezer E, Y. Sözmen E. Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS/MS. EJM. 2024;63:1–12.

MLA

Canbay, Erhan, et al. “Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS MS”. Ege Tıp Dergisi, vol. 63, no. 1, Mar. 2024, pp. 1-12, doi:10.19161/etd.1250567.

Vancouver

1.Erhan Canbay, Zeynep Çelik Canbay, Berrak Yeşilyurt, Serçin Doğan, İrem Arabacı, Ebru Demirel Sezer, Eser Y. Sözmen. Development of an Analytical Method For Determination of Urine 3-Phenylpyruvic Acid Based on Liquid-Liquid Extraction and LC-MS/MS. EJM. 2024 Mar. 1;63(1):1-12. doi:10.19161/etd.1250567