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Quantitative LC-MS: “How Certain Can we be!” Peter Stokes, Gavin O’Connor, Chris Mussell, & Ken Webb LGC Limited, Queens Rd. Teddington, Middlesex, TW11 0LY.
INTRODUCTION
THE ANALYSIS OF CREATININE IN HUMAN SERUM
CREATININE MEASUREMENTS USING DIFFERENT
Less than 20% of all LC-MS publications involve
MASS SPECTROMETRIC TECHNIQUES
quantitation. The reasons for the slow uptake of LC-MS for
quantitative analysis have been attributed to poor stability
LC-MS Extraction
GC-MS extraction
of atmospheric pressure ionisation and problems
associated with competing matrix reactions.
1. 1g Serum
1. 1g Serum
2. Add Hydrochloric acid (0.1M)
However, recent advancements in LC-MS technology such
2. Add labelled creatinine
as orthogonal spraying, the use of curtain or cone gases
3. Dilute in water
3. Equilibrate
and Z-SprayTM inlets have improved measurement precision
4. Add labelled creatinine
and the use of tandem mass spectrometry have helped to
4. Precipitate Protein (3mL ethanol)
5. Equilibrate
overcome many problems associated with matrix
5. Centrifuge 15mins, 400rpm
6. Remove creatine using ion
interference’s.
6. Evaporate to dryness @ 60°C
exchange
GC-MS has traditionally been the tool of choice for high
7. Reconstitute in mobile phase
7. Remove creatinine
accuracy measurements because measurements typically
8. Run on LC-MS
8. Evaporate to dryness
have very low CV’s.
9. Derivatise with MSTFA and
Concent 18.5
When comparing GC-MS with LC-MS it is important to
pyridine
consider the extraction AND measurement process. LC-MS
10. Run on GC-MS
methods are often much simpler than GC-MS methods and
although the final measurement may be less precise,
simplification of the extraction method means that results
generated by the two techniques are more comparable
than one might think.
Extraction time ~ 3 hours
Extraction time ~ 7 hours
EXACT MATCHING ISOTOPE DILUTION MASS
QUANTITATION SUMMARY USING LC-MS WITH
MEASUREMENT UNCERTAINTY
SPECTROMETRY (EM-IDMS)
DIFFERENT CALIBRATION REGEIMES
In this form of IDMS samples and standards are prepared
The amount of creatinine in the sample is calculated
gravimetrically to contain equimolar concentrations of
analyte and labelled analogue
W = the concentration of creatinine in sample W = the concentration of natural creatinine solution used to prepare the calibration blend m = mass of the natural creatinine standard added to the calibration blend; This results in both sample and standard being
m = mass of the labelled creatinine standard added to the calibration blend; indistinguishable on a mass spectrometer
m = mass of the labelled creatinine standard added to the sample blend; R’ = measured ratio of the sample blend; = Average measured ratio of the calibration blend injected before and after the sample Advantages:
The measurement uncertainty is defined by:
Excellent accuracy and precision
Uncertainty equation includes all components of
concentration calculation
c = concentration of creatinine in the standard solution ⎝ y ⎠ ⎝ z ⎠ ⎝ yc ⎠ Low uncertainty
Concent 18
m = mass of the creatinine-d3 added to the sample blend Disadvantages:
m = mass of creatinine used to prepare the calibration blend m = mass of creatinine-d3 added to the calibration blend Iterative process – need to repeat several times
Gravimetric preparation is time consuming
Uncertainty budget for the analysis of creatinine using EM-IDMS
Requires large amount of instrument time
Reduction of Systematic Errors in Quantitative Analysis by Isotope Dilution Mass Spectrometry (IDMS): An Iterative
Method; Henrion, A., Fresenius J. Anal. Chem., 1994, 350, 657-658
Guideline for Achieving High Accuracy in Isotope Dilution Mass spectrometry (IDMS); edited by M. Sargent, C.
Harrington & R. Harte. Published by RSC (UK) 2002.
OTHER HIGH ACCRACY LC-MS(MS) APPLICATIONS
Conclusion
The results shown in this presentation show that LC-MS
Ethynylestradiol in Drinking Water
can be an excellent tool for high accuracy applications and
Cholesterol in serum*
Level 1 (~2ng/g)
with the correct choice of calibration regime the results
obtained can be comparable with more traditional forms of
Lin. cal. solvent standards
1.1ng/g ± 0.66
mass spectrometry such as GC-MS.
1.7840mg/g ± 0.0076 (GC-MS)
Lin cal. matrix standards
2.21ng/g ± 1.03
1.7870mg/g ± 0.0059 (LC-MS)
Lin. cal. Matrix std’s Ethynylestradiol-d4 IS 2.06ng/g ±0.24
ACKNOWLEDGEMENTS
Lin. cal. Matrix std’s Estradiol-d4 IS
2.07ng/g ±0.52
2.7180mg/g ± 0.0056 (GC-MS)
Mike Welch - NIST
EM-IDMS 1.98ng/g
2.7137mg/g ± 0.0082 (LC-MS)
Andre Henrion - PTB
Celine S. J. Wolff Briche - LGC
Level 2 (~20ng/g)
*Comparison of gas chromatography and liquid chromatography mass spectrometric
David Carter - LGC
measurements for high accuracy analysis of cholesterol in human serum by isotope
Lin. cal. solvent standards
23.84ng/g ± 3.10
dilution mass spectrometry. Celine S.J. Wolff Briche. David Carter and Kenneth S.
Webb. Rapid Commun. in Mass Spectrom. 2002; 16: 848 - 853.
The work described in this poster was
Lin cal. matrix standards
13.72ng/g ± 5.78
supported under contract with the
Lin. cal. Matrix std’s Ethynylestradiol-d4 IS 19.65ng/g ± 3.53
LSD in Urine
Department of Trade and Industry as
part of the Valid Analytical
Lin. cal. Matrix std’s Estradiol-d4 IS
20.07ng/g ± 3.21
1.568ng/g ± 0.083ng/g
Measurement programme.
EM-IDMS 19.94ng/g

Source: http://www.nmschembio.org.uk/dm_documents/Quant_poster_white_background_pdf_4-dtq.pdf