Sains Malaysiana 50(7)(2021): 1913-1920

http://doi.org/10.17576/jsm-2021-5007-07

Validasi Kaedah Kromatografi Cecair Berprestasi Tinggi (HPLC) Jenis Penukar Ion dan Perbandingannya terhadap HPLC Jenis Fasa Berbalik bagi Penentuan Jumlah Homosisteina

(Validation of Ion Exchange High Performance Liquid Chromatography (HPLC) Method and Its Comparison with Reverse Phase HPLC for Total Homocysteine Determination)

AFFANDI OMAR¹*, SITI ROZILAH ABDUL KADIR², SALINA ABDUL RAHMAN¹, FATIMAH DIANA AMIN NORDIN¹, BALQIS KAMARUDIN¹, NUR JANNAIM MUHAMAD¹, ROSNANI MOHAMED¹, NURUL ADIBAH ROZALI², NORZAHIDAH KHALID², MUHD IRFAN BUKHARI AHMAD NAZRI² & JULAINA ABDUL JALIL¹

¹Unit Metabolisme Terwaris, Pusat Penyelidikan Pemakanan, Metabolisme dan Kardiovaskular, Institut Penyelidikan Perubatan, Kompleks Institut Kesihatan Negara, Kementerian Kesihatan Malaysia, Jalan Setia Murni U13/52, Bandar Setia Alam, 40170 Shah Alam, Selangor Darul Ehsan, Malaysia

²Unit Biokimia, Pusat Diagnostik Khas, Institut Penyelidikan Perubatan, Kementerian Kesihatan Malaysia, Jalan Pahang, 50588 Kuala Lumpur, Wilayah Persekutuan, Malaysia

Received: 25 June 2020/Accepted: 1 December 2020

ABSTRAK

Kajian ini dijalankan untuk menilai kemampuan kaedah penentuan jumlah homosisteina dalam plasma dengan menggunakan kromatografi cecair berprestasi tinggi jenis penukar ion (HPLC-IEC) dan membandingkan kaedah ini dengan kit komersil yang menggunakan kromatografi cecair berprestasi tinggi jenis fasa berbalik (HPLC-RPC). Validasi kaedah bagi penentuan jumlah homosisteina menggunakan HPLC-IEC telah dilakukan mengikut panduan yang dikeluarkan oleh Jabatan Standard Malaysia. Sebanyak 57 sampel daripada pesakit yang dihantar untuk penentuan jumlah homosisteina telah diuji secara serentak untuk menentukan korelasi antara kedua kaedah tersebut. Hasil kajian ini mendapati bahawa validasi kaedah ini telah memenuhi keperluan ISO MS 15189 dan kelinearan sehingga 500 µM dengan pekali penentuan, r² ialah 0.999. Kepersisan yang dikaji pada aras normal (10 µM) dan aras berpenyakit/abnormal (60 µM) bagi kebolehulangan adalah masing-masing 3.2% dan 3.8% manakala bagi kebolehasilan semula masing-masing adalah 8.0% dan 8.3%. Had pengesanan dan had pengiraan yang diperoleh adalah 0.9274 dan 3.0912 µM. Keputusan ketepatan bagi kembalian semula adalah 92% dan bias adalah -8.6%. Perbandingan kaedah yang telah divalidasi dengan kit komersil menunjukkan hubungan korelasi yang kuat antara kedua-dua kaedah ini (y = 0.721x + 4.034; r = 0.978; p<0.05). Oleh yang demikian, kaedah penentuan jumlah homosisteina menggunakan HPLC-IEC menunjukkan kepersisan yang memuaskan dan korelasi yang kuat setara dengan kit komersil yang dianalisis menggunakan HPLC-RPC. Kaedah ini lebih menjimatkan kos reagen dan mampu menganalisis lebih banyak sampel jika dibandingkan dengan kit komersil. Ini sekaligus dapat membantu dalam diagnosis bagi penyakit kepincangan metabolisme terwaris (IEM) terutama dalam metabolisme homosisteina dengan tepat.

Kata kunci: Jumlah homosisteina; kromatografi cecair berprestasi tinggi; validasi kaedah

ABSTRACT

The aim of this study was to validate the performance of plasma total homocysteine method using high performance of liquid chromatography ion exchange column (HPLC-IEC) and to compare this method with a commercially available kit analyzed using HPLC reverse phase column. Method validation was performed according to the Standards Malaysia Guidelines. Correlation study was done simultaneously between both methods using patients’ samples (n = 57) sent for total homocysteine determination. This study found that the HPLC-IEC method validation has met the MS ISO 15189 requirement criteria with linearity up to 500 µM (coefficient of determination, r² = 0.999). Precision was studied on normal (10 µM) and abnormal (60 µM) level, with 3.2 and 3.8% for repeatability and 8.0 and 8.3% for reproducibility.  The limit of detection and quantitation were 0.9274 and 3.0912 µM. As for accuracy, the results for recovery and bias were 92 and -8.6%. Method comparison showed a strong correlation between both methods (y = 0.721x + 4.034; r = 0.978; p<0.05). In conclusion, total homocysteine method using HPLC-IEC showed acceptable precision with strong correlation with commercial kit analyzed using HPLC-RPC. This method is more economical compared with commercial kit and it helps to diagnose inborn errors of metabolism (IEM) disorders especially in homocysteine metabolism accurately.    

Keywords: High performance liquid chromatography; method validation; total homocysteine

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*Corresponding author; email: fendi.omar@moh.gov.my