Increasing liquid chromatography-tandem mass spectrometry throughput by mass tagging: a sample-multiplexed high-throughput assay for 25-hydroxyvitamin D2 and D3.
BACKGROUND
The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography-tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D2 and D3 [25(OH)D2 and 25(OH)D3], which increased sample throughput 5-fold.


METHODS
After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D2 and 25(OH)D3 for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay.


RESULTS
TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 μg/L, 25(OH)D2], 2.2 nmol/L [0.9 μg/L, 25(OH)D3], and 10 nmol/L [4 μg/L, 25(OH)D2], 5 nmol/L [2 μg/L, 25(OH)D3], respectively. The assay was linear to 250 nmol/L (100 μg/L). Interassay CVs across the reportable range were 3.7%-15.2%. Spiked recoveries were 94%-119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D2 (n=1733) and 25(OH)D3 (n=7614) (R2=0.96 and 0.97), respectively.


CONCLUSIONS
Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D2 and 25(OH)D3 allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA.
Brian C
BC
Netzel
Kendall W
KW
Cradic
Eric T
ET
Bro
Adam B
AB
Girtman
Richard C
RC
Cyr
Ravinder J
RJ
Singh
Stefan K G
SK
Grebe
D015652:25-Hydroxyvitamin D 2 / Q000097:blood* / Q000737:chemistry; D001331:Automation; D002112:Calcifediol / Q000097:blood* / Q000737:chemistry; D002138:Calibration; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D006801:Humans; D015394:Molecular Structure; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards
D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol