1. RASHTRASANT TUKADOJI MAHARAJ NAGPUR UNIVERSITY, NAGPUR
DEVELOPMENT AND VALIDATION OF AN HPTLC METHOD FOR DETERMINATION OF METHOTREXATE IN HUMAN SERUM BY
Sonali G. Thorat
DEPARTMENT OF PHARMACEUTICAL SCIENCES
RASHTRASANT TUKADOJI MAHARAJ
NAGPUR UNIVERSITY, NAGPUR
INDIA

2. Introduction
Therapeutic Drug Monitoring (TDM)
TDM started out in 1970
TDM is broadly applied to;
Cardiovascular Agents,
Antiepileptics,
Antibiotics,
Respiratory Smooth Muscle Relaxants,
Cancer Chemotherapeutic Agents,
Immune-suppressants,
Antidepressants

3. Introduction
Antineoplastic agents satisfy the criteria for TDM;
Vastly variable pharmacokinetics
Narrow therapeutic indices
Sub-optimal therapy can greatly reduce the probability of cure for curable cancers.
Side effects, such as myelosuppression, can be life-threatening and may be related to higher than optimal therapy.

4. Literature Survey Methotrexate (MTX) Category: Breast, head and neck,
Leukaemia, lymphoma, lung, 
Osteosarcoma, bladder, and
Trophoblastic neoplasms.
Dose: Osteosarcoma Initial recommended dose: 12 g/m2 as a 4-hr infusion Choriocarcinoma Adult: 15-30 mg daily for 5 days Breast cancer Adult: 10-60 mg/m2 often with cyclophosphamide
and fluorouracil.
Lethal dose (LD50): 135 mg/Kg or 798 mg/m2
ADR: Bone marrow depression, hepatotoxicity, renal failure.
T1/2 = 8-15 h in higher doses; 3-10 h in lower doses.

5. Therapeutic Drug Monitoring of Methotrexate
Need of TDM
Dose calibration
Patient with multiorgan failure
To detect possible toxicity
Paediatric oncology
Methods Available
HPLC
HPLC-MS
U-HPLC-ESI-MS/MS
Proposed Method
HPTLC

6. Chromatographic conditions
Stationary Phase : Silica Gel 60 F254 TLC Plate
Thickness : 200 m
Mobile Phase : Toluene : Methanol (7:3 v/v)
Mode of Application : Band
Band Width : 5 mm
Sample volume : 10 L
Separation Technique : Ascending
Development Chamber : Twin trough glass chamber,
10 × 10 cm
Saturation Time : 20 min with mobile phase and spotted plate
Migration Distance : 80 mm
Detection : UV Densitometric scanning
Scanning Mode : Absorbance/ reflectance
Scanning Wavelength : 261 nm
Slit Dimension : 4 × 0.45 mm
Temperature : 25  3°C

7. HPTLC Method Development
Densitogram of blank serum
Methotrexate and IS at LLOQ

8. Internal std and Quality control samples
Internal standard : Etravirine (500 µg/ml)
Calibration curve standard : Methotrexate (500 µg/ml)
Quality control samples :
Dilution of methotrexate for quality control samples
The spiking of dilution in serum for QC sample 
Stock conc. (µg/ml)
Stock Aliquot (µl)
Volume made up with diluent (µl)
Final conc.
(µg/ml)
Working solution ID
500 15
LLOQ 25
LQC 50
MQC 90
HQC
Stock conc. (µg/ml)
Stock Aliquot (µl)
Volume made up with diluent (µl)
Final conc.
(µg/ml)
Working solution ID
500 15
LLOQ 25
LQC 50
MQC 90
HQC

9. Procedure and optimization
Optimization of procedure for sample preparation

10. Validation Selectivity:
% interference = Peak area response at Rf of analyte in blank ×100/peak area response at Rf of analyte in LLOQ
% interference = Peak area response at Rf of IS in blank ×100/peak area response at Rf of IS in LLOQ
Result of selectivity for methotrexate Result of selectivity for IS
Sample no.
Response at Rf of methotrexate in blank serum
Response of drug in LLOQ
% response in blank serum 1
917.5
0.00 2
926.5 3
1012.3 4
901.6 5
936.2
Sample no.
Response at Rf of Etravirine in blank serum
Response of Etravirine in LLOQ
% response in blank serum w.r.t LLOQ 1
5495.2
0.00 2
6078.3 3
5813.8 4
6102.3 5
5482.5

11. Matrix Effect
The matrix factor at low and high QC level was calculated:
Result of matrix effect for methotrexate: LQC HQC
Result of matrix effect for Etravirine
LQC HQC
Sample no.
Unextracted sample
Extracted sample
Matrix factor
Mean
1818
0.82 SD
158.77
134.43
0.063
%CV
7.26
7.39
7.68
Sample no.
Unextracted sample
Extracted sample
Matrix factor
Mean
0.90 SD
97.93
191.84
0.031
%CV
1.64
3.56
3.44
Sample no.
Unextracted sample
Extracted sample
Matrix factor
Mean
6385.9
5963.1
0.92 SD
89.85
190.27
0.03
%CV
1.40
3.19
3.26
Sample no.
Unextracted sample
Extracted sample
Matrix factor
Mean
0.88 SD
89.10
177.89
0.027
%CV
1.36
3.04
3.06

12. Calibration curve
Calibration curve standards were prepared by spiking working solution of analyte in serum.
A single calibration curve
was used for individual
analyte studied in the
method validation and
for each analytical run.
The lowest standard on the
calibration curve was
considered as the lower
limit of quantitation (LLOQ).

13. Precision and Accuracy
LLOQ, LQC, MQC and HQC were prepared in four replicates and applied with calibration standard.
Precision and accuracy was evaluated mean, S.D, and %CV values were calculated.
LQC, MQC and HQC samples should be within ±15% of their respective nominal values and LLOQ sample should be within ±20% of their respective nominal values.
Nominal concentration (µg/ml)
LLOQ
150ng/band
LQC
250ng/band
MQC
500ng/band
HQC
900ng/band
Mean
129.29
213.79
449.78
839.14
S.D
5.55
12.19
13.61
12.52
%CV
4.29
5.70
3.02
1.49

14. Sensitivity
To determine the lowest concentration that will be measured with an acceptable limit of accuracy and precision.
Four replicates of LLOQ samples were extracted.
The LLOQ concentration were calculated and the mean standard deviation, %CV and % accuracy were determined.
% Accuracy should be within %.
Sr.no
Calculated concentration
at LLOQ (ng/band)
% Accuracy
Mean
129.91
86.61
S.D
6.63
4.42
%CV
5.10

15. Recovery
Sr. No.
Unextracted sample LQC
Extracted sample LQC
Recovery
Mean
82.22
S.D
100.68
160.36
8.39
%CV
4.36
8.47
10.20
Unextracted sample MQC
Extracted sample MQC
87.92
94.57
111.09
1.61
2.68
3.58
1.83
Unextracted sample HQC
Extracted sample HQC
5433.3
88.69
137.8
157.46
1.68
2.37
2.89
1.89
Recovery experiment was performed in four replicates at LQC, MQC and HQC level for analyte along with internal standard.
The analytical results for extracted samples at three concentrations (equivalent to LQC, MQC and HQC) were compared with unextracted samples of concentrations equivalent to the extracted samples.

16. Stability
Stability study of methotrexate under various storage conditions (n=4).
Condition
Concentration (ng/band)
% stability
8 hr study
150
500
900
87.93
92.21
94.77
Three freeze-thaw
97.50
85.87
83.69
-40°C, 21 days
81.72
84.80
90.32

17. Application for TDM Patient 1:
Drug administered twice weekly by intrathecal route.
Serum samples obtained after 24, 48 and 72 h
Patient 2:
Parameter
Patient 1 (15mg)
Patient 2 (10mg)
t1/2 (h)
14.24
44.71
Cmax (ng/mL)
AUC0→t (ng/mL h)
CL (l/hr)
5.55
1.4

18. Patient Sample : Densitogram
Patient 1: Samples were collected by after drug administration, dose was 15 mg.
Patient 2: Samples were collected by after drug administration, dose was 10 mg.
Patient 1: 48 h after
MTX dosing spiked with IS
Patient 2: 48 h after
MTX dosing spiked with IS

19. Discussion Calibration curve range : 150 to 1000 ng/band
Selectivity: LLOQ > 20 %
Precision and Accuracy: LLOQ > 4.29 %
LQC, MQC and HQC > 5.70 , 3.02 and 1.49 % respectively
Sensitivity : LLOQ > % and Accuracy : 5.10 %
Recovery : LQC > 10.20, MQC > 1.83, HQC > 1.89
Samples were stable; in accordance to US-FDA guidelines

20. Discussion
This validated method was applied for TDM of two cancer patients on methotrexate therapy.
From the results we found that patient 1 showed normal values but in patient 2 values were abnormal.
The cause for abnormality was found to be liver and kidney dysfunction in patient 2.

21. Conclusion
The developed method was found to be useful in determination of methotrexate in serum samples.
This method was relatively simple, accurate and precise.
It can be applied in the routine clinical practice such as therapeutic monitoring of various drugs.

22. Acknowledgement
Anchrome Pvt Ltd, Mumbai, India
Rashtrasant Tukadoji Maharaj Regional
Cancer Hospital, Nagpur, India
Advanced Centre for Treatment Research
and Education in Cancer (ACTREC),
TATA Cancer Hospital, Mumbai

23. Thank you