Method for Determination of Leflunomide Metabolite over a 40,000-Fold Dynamic Range

Approximately, 1 % of the general population suffers from rheumatoid arthritis (RA) with three times greater prevalence in women. Leflunomide (ARAVA®) is a prodrug used for the treatment of RA. It is converted almost entirely to an active metabolite, teriflunomide, in the body. (Teriflunomide has been separately approved, as AUBAGIO®, for treatment of multiple sclerosis.) The metabolite acts as an immunomodulator and may aid in lessening the pain and progression of RA. Typical doses of leflunomide are 20-60 mg daily. The metabolite has a long half-life of approximately 14 days due to enterohepatic circulation. Therapeutic concentrations are in the range of 8 – 60 ug/mL but due to concerns of teratogenicity it is recommended that the drug be cleared to concentrations below 20 ng/mL plasma, for both men and women, prior to conceiving offspring. Consequently, quantitative analysis of serum/plasma samples must cover a range from low ng/mL up to 100 ug/mL or more. To cover this broad quantitative range for routine patient analysis we devised a strategy, described here, where the quantitative range is divided into two separate but overlapping regions. In this fashion we perform quantitation of this drug across a 40,000 fold concentration range.

Thanks are given to the ARUP Institute for Clinical and Experimental Pathology for making this work possible.

• An accurate and precise method has been developed and validated for teriflunomide.

• A very broad quantitative range is accommodated by splitting the concentration curve into two overlapping segments.

• The importance of conducting thorough anti-coagulant and matrix effect studies is emphasized.

• Qualitatively, lipemic, icteric, and hemolysed samples do not appear to produce significantly increased ionization suppression compared with a control matrix.

Teriflunomide: Scatter Plot

Deming Regr 1:1 Line

ARUP LC/MS/MS (ng/mL)100,00075,00050,00025,0000

NM

S L

C/M

S/M

S (

ng

/mL

)

100,000

75,000

50,000

25,000

0

Icteric, lipemic and hemolysed matrix types were prepared with appropriate additives for each situation. Specifically, mixed isomers of bilirubin were added to control matrix as a representation of icterus (200 ug/mL); Intralipid 20% was fortified into control matrix for 2.85% lipemia; hemolysed RBCs were added to create 6% hemolysed conditions. In the latter case, Hgb determinations ,made using a Beckman Coulter AcT- 10 Counter, and gave an Hgb concentration of 0.86 g/dL. The same experiments illustrated in table at right were repeated with icteric and hemolyzed conditions. Results were comparable to those shown here for lipemia.

XIC of -MRM : 269/ 160 Da

0.5 1.0 1.5 2.0 2.5 3.0

Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

Inte

nsi

ty,

cps

1.59

1.85

XIC of -MRM : 269/ 82 Da

leflunomide

teriflunomide

• A method was developed and validated to quantify the drug metabolite teriflunomide in human serum and plasma across the range of 5 ng/mL to 200 ug/mL.

• Calibrators were broken into two separate regions, with partial overlap, to accommodate the broad range necessary.

• Matrix effects were evaluated carefully in several collection tube types and with special matrix types of icteric, lipemic and hemolysed conditions.

• Freeze/ thaw stability was demonstrated through 3 additional cycles

• Storage stability was shown for 9 days at RT, 17 days at 4° C and z days at -20 ° C.

• Stock solution stability (10 mg/mL in dimethyl sulfoxide) was shown for 139 days.

TIC of -MRM (4 pairs): from Sample 12 (Red) of DataSET1.wiff (Turbo Spray) Max. 1.2e6 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

In

ten

sit

y,

cp

s

2.17

Serum, Heparin, EDTA NaF/K Oxalate

6 min Cycle time

XIC of -MRM (4 pairs): 273.000/82.000 Da from Sample 5 (Red) of DataSET1.wiff (Turb... Max. 1.1e6 cps.

0.5 1.0 1.5 2.0 2.5 3.0

Time, min

0.00

1.00e5

2.00e5

3.00e5

4.00e5

5.00e5

6.00e5

7.00e5

8.00e5

9.00e5

1.00e6

1.08e6

Inte

nsi

ty,

cp

s

1.57

Serum, heparin, EDTA

NaF/K Oxalate

4 min Cycle time

• The majority of patient samples fall in the range of the upper calibration curve (above). If a sample falls below the LOQ of the upper curve then the lower calibration curve is used.

Theoretical Grand Mean Mean % Within Between Total

ug/mL ug/mL Accuracy CV% CV% CV%

Lower

Controls Control A 0.02 0.020 101% 4.77 3.61 5.98

Control B 0.1 0.095 95.2% 1.90 2.79 3.38

Control C 0.8 0.771 96.4% 2.54 1.71 3.06

Upper

Controls Control D 1 0.982 98.2% 4.57 6.75 8.15

Control E 80 77.0 96.3% 3.80 2.53 4.56

Control F 170 161 94.6% 4.65 1.14 4.79

Lower Curve Back Calculated Accuracy

Calibrator (nominal conc.) Run 5 Run 7 Run 8 Run 11 Run 12 MEAN

Standard 1- 0.005 ug/mL 98.5 98.6 100 100 99.1 99.24

Standard 2 - 0.02 ug/mL 107 106 98.8 98.2 104 103

Standard 3 - 0.1 ug/mL 96.7 100 105 101 96.8 100

Standard 4 - 0.5 ug/mL 102 99.8 102 103 104 102.2

Standard 5 - 1.0 ug/mL 95.8 95.9 94.5 97 95.9 95.8

Upper Curve

Calibrator (nominal conc.) Run 13 Run 14 Run 15 Run 16 Run 17 MEAN

Standard 6 - 0.8 ug/mL 98.2 99.8 99.7 98.2 97.6 98.7

Standard 7 - 2 ug/mL 104 100 101 103 105 103

Standard 8 - 10 ug/mL 102 103 101 106 107 104

Standard 9 - 50 ug/mL 97.8 100 99.2 96.7 99.7 98.7

Standard 10 - 200 ug/mL 97.5 97.2 99.4 96.0 91.2 96.3

• The method uses simple protein precipitation.

• Separation is performed on a pentafluorophenyl phase.

• Supernatant from the upper curve analysis is diluted 100x before injection while lower curve is injected without dilution.

An original run time with lengthier column washing produced the results below and in tables at right. Shortening the injection cycle to 4 minutes yielded a broadened, slightly early eluting peak ,with gray topped (NaF/K Oxalate) tubes (below right).

KF/ Na Oxalate LLOQ EDTA LLOQCalc. conc. % Theor Quan/Qual Calc. conc. % Theor Quan/Qual

lot 1 0.00509 102 1.04 lot 1 0.00508 102 1.02

lot 2 0.00517 103 1.06 lot 2 0.00483 96.5 0.977

lot 3 0.00525 105 1.04 lot 3 0.00500 100 0.985

lot 4 0.00513 103 1.03 lot 4 0.00513 103 1.02

lot 5 0.00494 98.8 0.979 lot 5 0.00490 98.0 0.953

lot 6 0.00485 96.9 0.955 lot 6 0.00489 97.8 0.987

mean 0.00507 101 1.02 mean 0.00497 100 0.99

std dev 0.000149 std dev 0.000118

CV 2.95% CV 2.37%

Heparin LLOQ Serum LLOQCalc. conc. % Theor Quan/Qual Calc. conc. % Theor Quan/Qual

lot 1 0.00500 100 0.967 lot 1 0.00516 103 1.04

lot 2 0.00484 96.8 0.922 lot 2 0.00475 95.1 0.973

lot 3 0.00496 99.3 0.957 lot 3 0.00513 103 1.01

lot 4 0.00513 103 1.04 lot 4 0.00491 98.2 1.01

lot 5 0.00482 96.4 0.986 lot 5 0.00504 101 0.951

lot 6 0.00482 96.4 1.00 lot 6 0.00485 97.0 1.01

mean 0.00493 98.7 0.98 mean 0.00497 99.6 1.00

std dev 0.000125 std dev 0.000163

CV 2.54% CV 3.28%

Inte

nsi

ty, c

ps

Time, min 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2 6.6

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

Inte

nsi

ty, c

ps

0.5 1.0 1.5 2.0 2.5 3.0 Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

1.85

In recent studies we have been examining the influence of the same three matrix types on signal suppression by tee infusion. The two panels below show the relative intensity obtained from solvent injection (blue), bilirubin spiked into control matrix (green) and a pooled icteric samples with an I-index of 40 (red). Extraction is by protein precipitation.