Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum: A new approach

Ghassabian, Sussan; Rethwan, Nur Syazwani Ahmad; Griffiths, Lyn R.; Smith, Maree T.

doi:10.1016/j.jchromb.2014.09.032

Cited by 21 publications

(11 citation statements)

References 23 publications

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“…We have used this method by some modifications to measure homocysteine and pyridoxal 5-phosphate in samples of human serum and whole blood, respectively [16,17]. The matrix was first spiked with different concentrations of the analytes, and the endogenous concentrations of the analytes were estimated using the negative x-intercept of the calibration line.…”

Section: Methods Of Standard Additionmentioning

confidence: 99%

Linearity of Calibration Curves for Analytical Methods: A Review of Criteria for Assessment of Method Reliability

Moosavi¹,

Ghassabian²

2018

Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

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104

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Calibration curve is a regression model used to predict the unknown concentrations of analytes of interest based on the response of the instrument to the known standards. Some statistical analyses are required to choose the best model fitting to the experimental data and also evaluate the linearity and homoscedasticity of the calibration curve. Using an internal standard corrects for the loss of analyte during sample preparation and analysis provided that it is selected appropriately. After the best regression model is selected, the analytical method needs to be validated using quality control (QC) samples prepared and stored in the same temperature as intended for the study samples. Most of the international guidelines require that the parameters, including linearity, specificity, selectivity, accuracy, precision, lower limit of quantification (LLOQ), matrix effect and stability, be assessed during validation. Despite the highly regulated area, some challenges still exist regarding the validation of some analytical methods including methods when no analyte-free matrix is available.

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Section: Methods Of Standard Additionmentioning

confidence: 99%

Linearity of Calibration Curves for Analytical Methods: A Review of Criteria for Assessment of Method Reliability

Moosavi¹,

Ghassabian²

2018

Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

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104

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“…The C677T polymorphism was genotyped in the patient group in the GRC laboratory using previously published methods [ 9 ]. The Plasma homocysteine, folate, B 6 and B 12 levels were measured in an accredited pathology laboratory (TetraQ, University of Queensland, Brisbane) [ 17 , 18 ].…”

Section: Methodsmentioning

confidence: 99%

The effect of 1 mg folic acid supplementation on clinical outcomes in female migraine with aura patients

et al. 2016

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BackgroundMigraine is a common neurovascular condition that may be linked to hyperhomocysteinemia. We have previously provided evidence that reduction of homocysteine with a vitamin supplementation can reduce the occurrence of migraine in women. The current study examined the occurrence of migraine in response to vitamin supplementation with a lower dose of folic acid.MethodsThis was a 6 month randomised, double blinded placebo controlled trial of daily vitamin supplementation containing 1 mg of folic acid, 25 mg of Vitamin B6 and Vitamin B12, on reduction of homocysteine and the occurrence of migraine in 300 female patients diagnosed with migraine with aura.ResultsVitamin supplementation with 1 mg of folic acid, did not significantly decrease homocysteine levels (P = 0.2). The treatment group did not show a significant decrease in the percentage of participants with high migraine disability, severity or frequency at the end of the 6 month intervention (P > 0.1).Conclusion1 mg of folic acid in combination with vitamin B6 and B12 is less effective in reducing migraine associated symptoms compared to the previously tested dosage of 2 mg folic acid in combination with 25 mg of vitamin B6 and 400 μg of vitamin B12.

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“…Our method has now been successfully applied to the quantification of PLP in more than 600 samples of whole human blood collected from participants in a clinical trial, prior to and after a 6-month period of vitamin B6 supplementation. We have also successfully used the same approach for quantification of homocysteine, another endogenous compound in human serum, and the method has been validated successfully in accordance with the criteria specified in the 2012 EMEA guideline on bioanalytical method validation [18].…”

Section: Endogenous Plp Concentrationsmentioning

confidence: 94%

A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood

Ghassabian

Griffiths

Smith

2015

Journal of Chromatography B

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Quantification of pyridoxal-5'-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20μL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was >92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80°C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

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Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum: A new approach

Cited by 21 publications

References 23 publications

Linearity of Calibration Curves for Analytical Methods: A Review of Criteria for Assessment of Method Reliability

Linearity of Calibration Curves for Analytical Methods: A Review of Criteria for Assessment of Method Reliability

The effect of 1 mg folic acid supplementation on clinical outcomes in female migraine with aura patients

A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood

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