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INTRODUCTION

• HPLC with tandem mass spectrometric (MS/MS) detection is becoming increasingly common in clinical laboratories.

• The sensitivity and selectivity of HPLC/MS/MS allow accurate and precise quantification of parent drug in the presence of metabolites and other interferences.

• Currently, all quantitative HPLC/MS/MS Tacrolimus assays are “home brew” techniques requiring extensive development and validation before routine use.

• For some laboratories, this presents a major barrier to adoption. Furthermore, it contributes to lack of standardization which can be problematic when trying to compare results and performance.

• To overcome these limitations a reagent kit was developed based on a previously published method1.

• We now present the results of a multicenter trial of the first therapeutic drug monitoring reagent kit designed specifically for use with HPLC/MS/MS technology.

• The MassTrak Immunosuppressant Kit has been cleared by the FDA and is CE marked per 98/79/EC for the quantification of Tacrolimus (FK506; Prograf) in liver and kidney transplant patient whole blood samples as an aid in the management of Tacrolimus therapy.

Figure 1: The MassTrakTM Immunosuppressants TDM Kit.

Multicenter Validation of the MassTrakTM Reagent Kit for the Quantification of Tacrolimus in Whole Blood Using HPLC/MS/MS Donald P Cooper1, Kimberly L Napoli2, Paul J Taylor3, Catherine Hammett-Stabler4, Kendon S Graham1, Gareth W Hammond1, Michael E Franklin3, Webb S Lowe4, Quynhmai Nguyen2 and Michael R Morris1 1Waters Corporation, Manchester, United Kingdom; 2University of Texas Medical School at Houston, Houston, TX; 3Princess Alexandra Hospital, Brisbane, Australia; 4University of North Carolina Hospitals, Chapel Hill, NC.

OVERVIEW

• Validation of a new reagent kit for the LC/MS/MS analysis of Tacrolimus in whole blood is reported.

• The kit contains calibrators, controls, internal standard, chromatography column and detailed methods for sample preparation and analysis.

• The performance characteristics were found to be acceptable for the intended use of quantification of Tacrolimus (FK506; Prograf) in liver and kidney transplant patient whole blood samples as an aid in the management of Tacrolimus therapy.

Figure 2: Sample pre-treatment using the MassTrak Reagents.

Tacrolimus Concentration

Identity lineY = X

y = 1.02x + 0.0124

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Figure 4: Passing-Bablok analysis of the results for 71 Tac-rolimus International Proficiency Testing samples (www.bioanalytics.co.uk) determined using the MassTrak kit compared to the method mean for the HPLC group of laborato-ries subscribed to the scheme. The 95% CI for the slope and intercept were 1.0 to 1.04 and –0.17 to 0.20, respectively.

Accuracy

Linearity

RESULTS

• Within run and total imprecision evaluated over 20 days at three separate test centers were <10% over the range 2.0—29.9ng/mL (Table 3).

• When compared to independent, validated HPLC/MS/MS methods at three test centers using a minimum of 50 samples from liver transplant patients and 50 samples from kidney transplant patients (ie >300 individual samples) the MassTrak reagent kit provided results that differed by ≤10% at the lower and upper medical decision points (Table 4 & Figure 3).

• Accuracy was assessed by analyzing Tacrolimus IPT samples (71) at two centers. All results were within the acceptable limits of the scheme (mean +/- 3 SD). Passing-Bablok analysis gave a slope and intercept that were not statistically significantly different than 1.0 and 0, respectively (Figure 4).

• The assay was linear over the range 1ng/mL—31ng/mL (Figure 5).

• Mean recovery from drug-free whole blood and from spiked patient samples was between 97% and 109% (Data not shown).

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Figure 5: Example linearity data generated using CLSI EP6-A4. A “low” and “high” patient sample were mixed in defined pro-portions to create a sequence of 9 samples with Tacrolimus concentrations known to be related linearly. Polynomial regres-sion coefficients for 2nd & 3rd order curve fits were not signifi-cantly different from zero, indicating linearity. The plot shows a linear fit (N=5) with 95% confidence interval.

Table 3: Within run and total precision assessed with pooled patient samples analysed twice per day for 20 days as per CLSI EP5-A2 2.

Precision

Test Center

Tacrolimus Low Medium High

Houston

Concentration (ng/mL) 2.8 9.0 20.0

Within Run CV 3.4% 2.7% 1.9%

Total CV 4.7% 3.4% 3.6%

Brisbane

Concentration (ng/mL) 2.6 11.3 27.8

Within Run CV 5.7% 3.5% 2.4%

Total CV 7.9% 5.1% 2.7%

Chapel Hill

Concentration (ng/mL) 2.0 10.9 29.9

Within Run CV 5.6% 3.2% 2.4%

Total CV 7.6% 3.7% 3.7%

CONCLUSIONS

• The performance characteristics of the MassTrak™ reagent kit are acceptable for the routine therapeutic drug monitoring of Tacrolimus in kidney and liver transplant recipients7.

• The reagent kit may provide the potential for harmonization of results between laboratories using HPLC/MS/MS.

• The MassTrak kit provides a turn-key solution for those laboratories wanting to analyse Tacrolimus in kidney and liver transplant patients using HPLC/MS/MS.

Analyte MS 1

(m/z) MS 2

(m/z) Cone

Voltage (V)

Collision Energy

(eV)

Tacrolimus 821.5 768.5 28 22

Ascomycin 809.5 756.5 28 22

Table 2: Typical optimised MS/MS Settings for the analysis of Tacrolimus and the internal standard, Ascomycin.

Time (min) %A %B Flow (mL/min) Curve

0 50 50 0.6 1

0.4 0 100 0.6 11

1.0 50 50 0.6 11

Table 1: Step gradient used to elute the MassTrak TDM col-umn. A=water and B=methanol, both containing 2mM ammo-nium acetate and 0.1% formic acid.

METHODS

• Whole blood calibrators, quality controls and internal standard (Figure 1) were prepared by redissolving in the appropriate solvent as described in the MassTrak user manual.

• Calibrators, QCs and patient samples were prepared for analysis as outlined in Figure 2.

• Samples were analysed using the MassTrak TDM C18 2.1mm x 10mm column eluted with a step gradient (Table 1).

• Tacrolimus and Ascomycin (internal standard) were detected using a Quattro micro tandem mass spectrometer operated in MRM mode (Table 2).

• Tacrolimus concentrations in test samples were determined by reference to the calibration curve.

The following assay characteristics were assessed at three evaluation centres using protocols based on the appropriate CLSI2-4 and US FDA5 guidelines: precision, accuracy, linearity, recovery and method comparison.

Method Comparison

Table 4 & Figure 3: Deming regression analysis of pooled liver & kidney method comparison data from each of the three evaluation centers. Each patient sample was analysed in duplicate using the MassTrak method and a validated comparative HPLC/MS/MS method according to Reference 3. Upper & lower medical decision points of 5ng/mL and 15ng/mL were selected based on the current Tacrolimus monitoring consensus document6.

Houston: Pooled Liver & Kidney Data

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Brisbane: Pooled Liver & Kidney Data

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UNCH: Pooled Liver & Kidney Data

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Deming Regression

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Medical Decision Point

Test Center N Deming Regression

(value ± 95% CI) Medical Decision Point

5ng/mL (95% CI) Medical Decision Point 15ng/mL (95% CI)

Houston 109 Slope 1.092 (1.080 to 1.104)

5.3 (5.2 – 5.4)

16.2 (16.1 – 16.4)

Int. -0.165 (-0.266 to -0.065) R 0.9965

Brisbane 100 Slope 1.107 (1.081 to 1.133)

5.4 (5.3 – 5.5)

16.5 (16.2 – 16.7)

Int. -0.148 (-0.357 to 0.061) R 0.9862

Chapel Hill 100 Slope 0.993 (0.974 to 1.012)

4.9 (4.8 – 5.0)

14.8 (14.7 – 15.0)

Int. -0.06 (-0.21 to 0.09) R 0.9907

1. B G Keevil, S McCann, D P Cooper & M R Morris. “Evaluation of a rapid micro-scale assay for Tacrolimus by liquid chromatography-tandem mass spectrometry”. Annals of Clinical Biochem. 39, 487-492, 2002.

2. CLSI Evaluation of Precision Performance of Quantitative Measurement Methods, EP5-A2, August 2004.

3. CLSI Method Comparison and Bias Estimation Using Patient Samples, EP9-A2, September 2002.

4. CLSI Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach, EP6-A, April 2003.

5. Class II Special Controls Guidance Document: Cyclosporine and Tacrolimus Assays; Guidance for Industry and FDA, September, 2002.

6. M Oellerich, V W Armstrong, E Schutz, & L M Shaw “Theraputic Drug Monitoring of Cyclosporine and Tacrolimus. Clin. Biochem. 31, 309 – 316, 1998.

7. L M Shaw, T M Annesley, B Kaplan & K L Brayman. “Analytic Requirements for Immunosuppressive Drugs in Clinical Trials” Ther. Drug Monitoring, 17, 577—583, 1995.

References

Figure 6: Extract prepared from drug-free whole blood was in-jected whilst the internal standard Ascomycin (A) or Tacrolimus (B) was infused post-column (red traces). In separate analyses, the elution positions of Ascomycin & Tacrolimus were determined (green traces). No significant matrix effect was seen at the elu-tion position of the analyte or internal standard.

Time0.50 1.00 1.50 2.00

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100ASCO_021204_4 Sm (Mn, 2x2) MRM of 1 Channel ES+

809.6 > 756.21.46e4

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100FK_021204_5 Sm (Mn, 2x2) MRM of 1 Channel ES+

821.6 > 768.21.97e4

0.92

A B

Matrix Effect (Ion Suppression)

• Post-column infusion studies demonstrated the lack of significant matrix effects (ion suppression / ion enhancement) at the elution positions of the analyte and the internal standard (Figure 6).