Determination and Pharmacokinetics Mitomycin C in Rat

Plasma by Liquid Chromatography-Mass Spectrometry

Ruth A
Introduction

• Iyer and Szybalski (1963) showed that mitomycin C activates alkylation and deoxyri-

bonucleic acid (DNA) cross-linking antiproliferative activity.

• MMC is currently used in various fields of the bladder,

colorectal, and stomach cancer.

• MMC works as an alkylating agent and DNA crosslinker,

which prevents DNA from being transcribed into RNA and,

thus, inhibits the synthesis of proteins

Introduction

• DNA's guanine nucleotide can form a covalent cross-link with an-

other guanine nucleotide when an alkylating chemical attaches an

alkyl group (CnH2n+1) to it.

• The DNA double helix is prevented from unwinding and separating

by the presence of covalent bonds between the strands, such as

those produced by MMC.\

• This research started while conducting a colorectal cancer research requested by Professor Park Eun-jung.
Objective
• Determination and validation of mitomycin C in rat plasma.

• Using this method, pharmacokinetic study of mitomycin C in rat.

Experimental
1. Sample preparation

2. Column and chromatographic condition

- Ionization mode : positive, MRM : 335.233 > 242.3

- Hypersil GOLD (Thermo scientific, 50 * 2.1 mm)

DP EP CEP CE CXP
36 V 3V 16 V 21 V 4 V

3. Validation

4. Pharmacokinetic study
Result Mass spectra
Result Representative chromatograms

MMC Blank plsma Spiking LOQ MMC IV 10min sample

Intensity, cps

CBZ 200 ng/mL CBZ 200 ng/mL

CBZ

Time (h)
* Validation 은 현재 진행중이지만 , PK 는 작년 5 월에 진행 . 하지만 , 지금 method 가 변경됨 .
Result Precision & accuracy
Mitomycin C
10 30 1000 4000
Day-1
Mean 8.62 34.5 982 4017
Precision 11.9 4.22 2.57 3.81
Accuracy 13.8 15.2 1.80 0.42
Day-2
Mean 8.44 35.8 951 4043
Precision 9.09 12.3 4.68 3.98
Accuracy 15.6 19.3 4.90 1.08

• Matrix effect = Post spiking analyte peak area/ Neat analyte peak area

• Recovery = Pre spiking analyte peak area/ Post analyte peak area
Result Matrix effect & Recovery

Concentration Mitomycin C
(ng/ml) Matrix effect Recovery

10 84 % 226 %
30 109 % 198 %
1000 71 % 249 %
4000 85 % 244 %
IS (Carbamazepine) 43 % 76 %

• Precision = (standard deviation of concentration / mean concentration) * 100

• Accuracy = │100 – (mean concentration * 100 / nominal concentration)│

Result Stability
LOQ (0.01 μg/ml) QCL (0.03 μg/ml) QCM (1 μg/ml) QCH (4 μg/ml)
Condition TEST
Mean SD Mean SD Mean SD Mean SD
Short-term 10.2 0.8 25.6 1.5 1167 90 3843 223
Freeze-thaw 9.8 1.1 25.7 0.6 1220 82 3787 93
Long-term
Post-preparative
Autosampler

• Short-term stability (at room temperature for 4 h)-Done

• Freeze-thaw (-20 °C, 3cycle)-Done

• Long-term stability (at -20 °C for 30 days)-Doing

• Post-preparative stability (4 °C, 7 day)-Doing

• Autosampler stability (at 10 °C for 24 h)-Doing

Result Pharmacokinetic study in rat.

 Plasma concentration versus time curve and PK parameter

10,000

IV
Conc (ng/ml)

PK parameter IV PO
1,000
PO
Cmax (ng/mL) - 50 ± 22

Tmax (hr) - 0.3 ± 1.4

100 Tλ1/2 (hr) 1.4 ± 0.1 0.7 ± 0.01

AUC0-8h (ng´hr/mL) 3594 ± 350 54 ± 22

AUC0-∞ (ng´hr/mL) 3640 ± 354 62 ± 25

10
CL (L/kg/hr) 1.38 ± 0.13

Vss (L/kg) 0.7 ± 0.1

BA, F (%) 1.5

1
0 2 4 6 8

Time (h)
 IV and PO administration (5 mg/kg) in Male 8 weeks SD rat (n=3).
Conclusion

• A new analysis method for MMC using LC-MS/MS was developed in rat plasma.

• Pharmacokinetic experiments were performed using this assay.

Further planned experiments
Validation 다시 진행 (LC-MS/MS) 수리 이후 진행 예정

다시 진행해야할 실험

- Precision & Accuracy, Matrix effect & Recovery, Stability