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 - - Chromatography Online
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
Volume 14, Issue 1

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.


Figure 1: Chromatogram representing 100 ng/mL 25-mono-hydroxy-vitamin D3 (red) and 25-mono-hydroxy-vitamin D2 (Blue) spiked in human plasma.
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.




Results


Table 1: Accuracy and precision of the QC samples.
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.

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.

Martin Sibum, Spark Holland bv, Emmen, The Netherlands.

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:




Spark Holland bv
P. de Keyserstraat 8, PO box 3887800 AJ Emmen, The Netherlands
E-mail:
Website: http://www.sparkholland.com/

ADVERTISEMENT

blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:




 

LCGC COLUMNISTS 2014

Sample Prep Perspectives | Ronald E. Majors:

LCGC Columnist Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments in existing technology lines.

LATEST: The Role of Selectivity in Extractions: A Case Study

History of Chromatography | Industry Veterans:

With each installment of this column, a different industry veteran covers an aspect of the evolution and continued development of the science of chromatography, from its birth to its eventual growth into the high-powered industry we see today.

LATEST: Georges Guiochon: Separation Science Innovator

MS — The Practical Art| Kate Yu:
Kate Yu is the editor of 'MS-The Practical Art' bringing her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers.

LATEST: Mass Spectrometry for Natural Products Research: Challenges, Pitfalls, and Opportunities


LC Troubleshooting | John Dolan:

LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan, Vice President of LC Resources and world renowned expert on HPLC, is able to highlight common problems and provide remedies for them.

LATEST: LC Method Scaling, Part I: Isocratic Separations

More LCGC Chromatography-Related Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>


Source: LCGC Asia Pacific,
Click here