Hironori Kobayashi (Presenter)
Shimane University Faculty of Medicine
Bio: My specialty is pediatric endocrinology and an inborn errors of metabolism. Now I am interested in development of for newborn screening (e.g. MPS, urea cycle disorders, SCID, X-ALD), and analysis of adrenal steroid.
Authorship: Hironori Kobayashi(1), Tomoyoshi Shiroshita(2), Kenji Yamada(1), Ryosuke Bo(1), Yuki Hasegawa(1), Toshimasa Ito(2), Akira Ideno(2), Toshinari Ohara(2), Seiji Yamaguchi(1)
(1) of pediatrics, Shimane University Faculty of Medicine , (2) SEKISUI MEDICAL Co., LTD
| Short Abstract Method for determination of acylcarnitine by LC-MS/MS was developed to improve existing techniques that are not suitable for the clinical diagnosis tests due to quantitative differences between analytical instruments and the lack of internal calibrator. The validation of AC determination assay in serum was conducted and the intra- and inter-day repeatability, accuracy, linearity, recovery and precision were confirmed. The previously developed method was simplified and precision was significantly improved by addition of the calibrator. We believe that this method has the potential to become standard method for clinical exam of positively screened infants or follow-up patients. |
Long Abstract
【Introduction】
Newborn screening (NBS) using MS/MS have become popular in worldwide. Analysis of acylcarnitines in dried blood spot (DBS) is performed on either NBS program or those patients who are suspected to be some inborn errors of metabolism. The demands for analysis of acylcarnitines (ACs) in serum have increased for the purpose of clinical exam in clinical practice.
Existing method for analysis of ACs was developed for both high throughput screening and low cost screening. In this method, we determine quantity by comparison chromatographic heights of internal standard and target material extracted from DBS. However, it’s not suitable for the clinical tests because of difficulty to keep quantitativity between analytical instruments, or of influence of ion suppression derived from matrix such as phospholipid in blood.
In this study, we developed and validated the new method for AC analysis for the purpose of clinical exam.
【Methods】
Standard master mix composed of 15 types of AC was serially diluted with ethanol containing 1% formic acid and added to pooled human serum sample previously subjected to dialysis or phosphate buffer solution including 5% fat free human serum albumin (5%HSA-PBS). 5 concentration standard curve and 2 concentration quality control samples were prepared.
Additionally, samples confirming recovery rate were prepared with non-dialyzed pooled human serum.
All previously prepared samples were supplemented with calibrator and after addition of ethanol sample was subjected to deproteination, dried up under the stream of nitrogen, re-dissolved, centrifuged and resulting supernatant subjected to analysis performed with LC-MS/MS（LC-20A：Simadzu and API2000：AB Sciex）and HILIC mode column, with 0.05% formic acid aq and 0.05% formic acid acetonitrile as mobile phase.
With the above method the intra- and inter-day repeatability, accuracy, linearity, recovery and precision were confirmed.
【Results and Implications】
In dialyzed pooled human serum samples linearity of AC was confirmed in the following quantiation ranges: Free carnitine (C0; 2.0 - 200μmol/L), Acetylcarnitine (C2; 1.0 - 100), Propionylcarnitine
(C3; 0.24-24), Butyrylcarnitine (C4; 0.1-10), Isovalerylcarnitine (C5; 0.06-6), (3-Hydroxyisovaleryl) carnitine (C5-OH; 0.1-10), Glutarylcarnitine (C5-DC; 0.05-5), Hexanoylcarnitine (C6; 0.05-5), Octanoylcarnitine (C8; 0.05-5), Decanoylcarnitine (C10; 0.05-5), Dodecanoylcarnitine (C12; 0.05-5), Tetradecanoylcarnitine (C14; 0.06-6), Tetradecenoylcarnitine (C14:1; 0.06-6), Palmitoylcarnitine (C16; 0.24-2.4) and Stearoylcarnitine (C18; 0.1-10), and the coefficient of correlation (r) of the calibration curve was 0.9942-0.9998.
In 5%HAS-PBS samples the quantitation ranges of all 15 types of AC were at the same levels as the dialyzed serum samples and the linearity with coefficient of correlation (r) at 0.9900-0.9999 was proved.
Intra-day repeatability (RE%) and accuracy (CV%) of serum samples was confirmed at CV% = 1.0-12.3% and RE% = -13.6-10.0%.
The 3 day inter-day tests results were between CV% = 2.4-10.2% and RE% = -11.5-8.1% .
Accuracy of the serum samples was confirmed with 5%HAS-PBS calibration curve samples and the CV% value ranged between 0.3% and 7.5%.
The repeatability of serum samples was not calculated due to endogenous effects of naturally occurring AC amounts in human serum.
Those results proved that since there is no difference in the precision samples between serum and 5%HAS-PBS samples measurement, both sample preparation methods can be used for AC determination interchangeably.
Repeatability (RE%) of the high and low concentration standard solution was at the acceptable range of RE% = -6.2-9.8% in all AC types, additionally the recovery of those samples was confirmed.
【Conclusions】
This method describes preparation and measurement of AC in human serum sample. The developed method was simplified and method precision was significantly improved by addition of the calibrator.
We believe that this method has the potential to become standard method for clinical exam of positively screened infants or follow-up patients.
References & Acknowledgements:
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