Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry

Petra Magdolna Molnár, L. Dux, Hans Reinauer, Michael Kress, Theodorus Akerboom, Edit Szederkényi, Patricia Kaiser

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Background: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required. Methods: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs +. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [ 2H 12]-Cyclosporin A [ 13C, 2H 3]-Rapamycin, [ 13C, 2H 2]-Tacrolimus, and [ 13C 2, 2H 4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards. Results: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8-102.5% for cyclosporin A, 101.6-103.0% for sirolimus, 100.0-101.2% for tacrolimus, and 99.5-102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17-2.60% for cyclosporin A in the concentration range between 8.1 and 979 μg/L, 0.92-1.72% for sirolimus in the concentration range between 2.1 and 33.2 μg/L, 0.44-1.06% for tacrolimus in the concentration range between 2.0 and 30.8 μg/L and 0.82-4.34% for everolimus in the concentration range between 2.1 and 31.4 μg/L. Conclusions: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.

Original languageEnglish
Pages (from-to)983-992
Number of pages10
JournalClinical Laboratory
Volume57
Issue number11-12
Publication statusPublished - 2011

Fingerprint

Liquid chromatography
Immunosuppressive Agents
Liquid Chromatography
Isotopes
Dilution
Mass spectrometry
Sirolimus
Mass Spectrometry
Blood
Tacrolimus
Cyclosporine
Pharmaceutical Preparations
formic acid
Transplantation (surgical)
Recovery
High Pressure Liquid Chromatography
High performance liquid chromatography
Liquids
Liquid-Liquid Extraction
Drug Monitoring

Keywords

  • Cyclosporin A
  • Everolimus
  • LC-ID-MS/MS
  • Reference measurement procedure
  • Sirolimus
  • Tacrolimus

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Molnár, P. M., Dux, L., Reinauer, H., Kress, M., Akerboom, T., Szederkényi, E., & Kaiser, P. (2011). Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry. Clinical Laboratory, 57(11-12), 983-992.

Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry. / Molnár, Petra Magdolna; Dux, L.; Reinauer, Hans; Kress, Michael; Akerboom, Theodorus; Szederkényi, Edit; Kaiser, Patricia.

In: Clinical Laboratory, Vol. 57, No. 11-12, 2011, p. 983-992.

Research output: Contribution to journalArticle

Molnár, PM, Dux, L, Reinauer, H, Kress, M, Akerboom, T, Szederkényi, E & Kaiser, P 2011, 'Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry', Clinical Laboratory, vol. 57, no. 11-12, pp. 983-992.
Molnár, Petra Magdolna ; Dux, L. ; Reinauer, Hans ; Kress, Michael ; Akerboom, Theodorus ; Szederkényi, Edit ; Kaiser, Patricia. / Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry. In: Clinical Laboratory. 2011 ; Vol. 57, No. 11-12. pp. 983-992.
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abstract = "Background: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required. Methods: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1{\%} v/v formic acid and 0.1 mmol/L Cs +. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [ 2H 12]-Cyclosporin A [ 13C, 2H 3]-Rapamycin, [ 13C, 2H 2]-Tacrolimus, and [ 13C 2, 2H 4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards. Results: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8-102.5{\%} for cyclosporin A, 101.6-103.0{\%} for sirolimus, 100.0-101.2{\%} for tacrolimus, and 99.5-102.4{\%} for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17-2.60{\%} for cyclosporin A in the concentration range between 8.1 and 979 μg/L, 0.92-1.72{\%} for sirolimus in the concentration range between 2.1 and 33.2 μg/L, 0.44-1.06{\%} for tacrolimus in the concentration range between 2.0 and 30.8 μg/L and 0.82-4.34{\%} for everolimus in the concentration range between 2.1 and 31.4 μg/L. Conclusions: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.",
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AU - Molnár, Petra Magdolna

AU - Dux, L.

AU - Reinauer, Hans

AU - Kress, Michael

AU - Akerboom, Theodorus

AU - Szederkényi, Edit

AU - Kaiser, Patricia

PY - 2011

Y1 - 2011

N2 - Background: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required. Methods: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs +. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [ 2H 12]-Cyclosporin A [ 13C, 2H 3]-Rapamycin, [ 13C, 2H 2]-Tacrolimus, and [ 13C 2, 2H 4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards. Results: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8-102.5% for cyclosporin A, 101.6-103.0% for sirolimus, 100.0-101.2% for tacrolimus, and 99.5-102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17-2.60% for cyclosporin A in the concentration range between 8.1 and 979 μg/L, 0.92-1.72% for sirolimus in the concentration range between 2.1 and 33.2 μg/L, 0.44-1.06% for tacrolimus in the concentration range between 2.0 and 30.8 μg/L and 0.82-4.34% for everolimus in the concentration range between 2.1 and 31.4 μg/L. Conclusions: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.

AB - Background: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required. Methods: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs +. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [ 2H 12]-Cyclosporin A [ 13C, 2H 3]-Rapamycin, [ 13C, 2H 2]-Tacrolimus, and [ 13C 2, 2H 4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards. Results: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8-102.5% for cyclosporin A, 101.6-103.0% for sirolimus, 100.0-101.2% for tacrolimus, and 99.5-102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17-2.60% for cyclosporin A in the concentration range between 8.1 and 979 μg/L, 0.92-1.72% for sirolimus in the concentration range between 2.1 and 33.2 μg/L, 0.44-1.06% for tacrolimus in the concentration range between 2.0 and 30.8 μg/L and 0.82-4.34% for everolimus in the concentration range between 2.1 and 31.4 μg/L. Conclusions: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.

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