Determination of 25-mono-hydroxy-vitamin D2 and D3 in Human Plasma Using a Symbiosis Pico System: ACN Free On-line SPE-LC–MS–MS
LCGC Asia Pacific
Spark Holland Application Note
Introduction
The method developed runs on a fully automated platform integrating solid-phase extraction (SPE) and LC (Symbiosis Pico) without any manual pretreatment step(s). Automatic pretreatment results in high throughput, low CVs and high precision. Sample preparation has been simplified to just one operator step before the system takes over the automated analysis of the samples.
Instrumentation
A Spark Holland Symbiosis PICO system with a Mistral column oven and an Applied Biosystems/MDS Analytical Technologies API 4000 LC–MS–MS system are used for this application. The API 4000 is equipped with a Heated Nebulizer (APCI) probe.
Symbiosis Pico is Spark Holland's unique (u)HPLC solution with integrated on-line SPE-LC–MS automation. The system offers large flexibility in processing different types of samples selecting one of the three fully automated operational modes LC; XLC (extraction liquid chromatography) and mXLC (multidimensional on-line SPE)
Experimental
Sample preparation consists of diluting a human sample with a special protein disrupting buffer, followed by mixing. Samples are then placed in the Symbiosis autosampler. 100 µL of sample is injected on a Spark HySphere C8EC-SE sorbent SPE cartridge for on-line sample clean-up and concentration.
The mobile phase consists of formic acid, water and methanol with separation on a Merck KGaA Chromolith SpeedROD RP-18e (endcapped), 50 × 4.6 mm HPLC column. An example chromatogram of the separation achieved is shown in Figure 1.
Figure 1: Chromatogram representing 100 ng/mL 25-mono-hydroxy-vitamin D3 (red) and 25-mono-hydroxy-vitamin D2 (Blue) spiked in human plasma.
Results
A calibration curve was created by injecting a full set of calibration standards ranging from 5 to 500 ng/mL spiked human plasma. The calibration curve is calculated with a R2 of 0.999 with a 1/X weighting for both compounds.
Table 1: Accuracy and precision of the QC samples.
Conclusion
This method was evaluated using spiked human sodium heparin plasma samples. The developed on-line SPE-MS–MS method has an absolute recovery of more then 90%, and calibration curve with a linear range from 5–500 ng/mL (R = 0.999 for both compounds). The developed method has an accuracy ranging from 89–117% and precision of 0.1–7.9 %CV. The estimated quantification limits for both analytes are more than sufficient to allow the analytical method to be used for therapeutic drug monitoring. When injecting 100 µL, containing 50 µL of plasma, a detection limit of 5 ng/mL is achieved.
References
1. Spark application note 0053-080-01.
2. Applied Biosystem Vitamin D i-method (User Contributed i-method by Spark).
For more information please send an e-mail to: Applications@sparkholland.com
Spark Holland bv
P. de Keyserstraat 8, PO box 3887800 AJ Emmen, The Netherlands
E-mail: Applications@sparkholland.com
Website: www.sparkholland.com
New Study Uses MSPE with GC–MS to Analyze PFCAs in Water
January 20th 2025Scientists from the China University of Sciences combined magnetic solid-phase extraction (MSPE) with gas chromatography–mass spectrometry (GC–MS) to analyze perfluoro carboxylic acids (PFCAs) in different water environments.
The Next Frontier for Mass Spectrometry: Maximizing Ion Utilization
January 20th 2025In this podcast, Daniel DeBord, CTO of MOBILion Systems, describes a new high resolution mass spectrometry approach that promises to increase speed and sensitivity in omics applications. MOBILion recently introduced the PAMAF mode of operation, which stands for parallel accumulation with mobility aligned fragmentation. It substantially increases the fraction of ion used for mass spectrometry analysis by replacing the functionality of the quadrupole with high resolution ion mobility. Listen to learn more about this exciting new development.
A Guide To Finding the Ideal Syringe and Needle
January 20th 2025Hamilton has produced a series of reference guides to assist science professionals in finding the best-suited products and configurations for their applications. The Syringe and Needle Reference Guide provides detailed information on Hamilton Company’s full portfolio of syringes and needles. Everything from cleaning and preventative maintenance to individual part numbers are available for review. It also includes selection charts to help you choose between syringe terminations like cemented needles and luer tips.
The Complexity of Oligonucleotide Separations
January 9th 2025Peter Pellegrinelli, Applications Specialist at Advanced Materials Technology (AMT) explains the complexity of oligonucleotide separations due to the unique chemical properties of these molecules. Issues such as varying length, sequence complexity, and hydrophilic-hydrophobic characteristics make efficient separations difficult. Separation scientists are addressing these challenges by modifying mobile phase compositions, using varying ion-pairing reagents, and exploring alternative separation modes like HILIC and ion-exchange chromatography. Due to these complexities, AMT has introduced the HALO® OLIGO column, which offers high-resolution, fast separations through its innovative Fused-Core® technology and high pH stability. Alongside explaining the new column, Peter looks to the future of these separations and what is next to come.
Oasis or Sand Dune? Isolation of Psychedelic Compounds
January 20th 2025Magic mushrooms, once taboo, have recently experienced a renaissance. This new awakening is partially due to new findings that indicate the effects of psilocybin, and its dephosphorylated cousin psilocin may produce long lasting results for patients who might be struggling with anxiety, depression, alcohol and drug abuse, and post-traumatic stress disorder. Hamilton Company has developed a methodology for the isolation and identification of 5 common psychedelic compounds used in the potential treatment of disease. The PRP-1 HPLC column resin remains stable in the harsh alkaline conditions ideal for better separations.
