Development and validation of a bioanalytical technique entitled

5-fluorouracil (5FU) used in therapeutic drug monitoring.

 

Bhakti Patil¹, Nitesh Chavan², Vikram Gota², Vaidhun Bhaskar¹, Preeti Kulkarni¹*

¹Department of Quality Assurance, Gahlot Institute of Pharmacy, Plot 59, Sector 14, Kopar Khairane, Navi Mumbai, Maharashtra 400709.

²The Advanced Centre for Treatment, Research and Education in Cancer (ACTREC),

Kharghar, Navi Mumbai.

 

ABSTRACT:

Aim: The primary goal of this research was to create a therapeutic drug monitoring (TDM) approach for the oldest anticancer medication, 5-fluorouracil (5FU). Materials And Methods: We employed a Plasma matrix for analyte extraction through protein precipitation. Chromatographic separation of the analyte with the internal standard, 5-bromouracil (5BU), was carried out using a Phenomenex Luna C18 150*4.6mm 5um analytical column with an isocratic program. Gimeracil, which serves as a DPD enzyme inhibitor, was used to enhance the stability of 5FU in blood. The calibration curve for 5-fluorouracil was linear (r2 ≥ 0.99) from a range of 0.2μg/ml to 10μg/ml. The validation of the method was conducted following the bioanalytical method guidelines outlined by ICH and FDA in 2022. Conclusion: The method has been successfully developed and validated as a therapeutic drug monitoring (TDM) service to accurately determine the level of 5FU concentration in patients with gastric conditions, ensuring the specificity, sensitivity, and reliability of the method.

 

 

 

INTRODUCTION: 

Therapeutic drug monitoring is the employment of analytical techniques to arbitrate medicament concentrations in plasma and to clarify the usage of the data to influence concentration in order to elaborate safe and efficient medicinal regimens. This process allows therapeutic concentrations of a drug to be achieved more quickly and safely than is possible with empirical dosing regimens.¹ Therapeutic drug monitoring helps to plan a dosage program suitable for the patient; it helps increase medication effectiveness, reduce drug toxicity, and for diagnostic purposes, individualize drug therapy.^2,3

 

 

Gastrointestinal cancer accounts for the highest number of deaths among individuals diagnosed with any type of cancer. It pertains to cancer that develops in the gastrointestinal tract (GI tract) and its associated organs involved in digestion, including the esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum, and anus.⁴

 

For more than half a century, 5-fluorouracil (5FU) has been essential in the chemotherapy treatment of cancer patients. 5FU has a substantial range of pharmacokinetic variation, resulting in significant differences in dosage within individual patients and effect which leads to its need of Therapeutic drug monitoring.⁵

 

 

Figure 1: Chemical Structure of 5Fluorouracil (5FU)⁶

5-Fluorouracil (5-FU) is widely recognized as an effective treatment option for various types of cancer, including breast, head and neck, gastrointestinal, and colorectal cancers. Despite its longstanding use in cancer therapy, 5-FU remains a standard drug due to its potency and effectiveness. This medication is a pyrimidine analog that replaces a hydrogen atom with a fluorine atom at the C-5 position. Upon entering the cells, 5-FU is converted into different active metabolites, such as fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP), and fluorouridine triphosphate (FUTP). These metabolites interfere with RNA synthesis and inhibit the activity of thymidylate synthase (TS), disrupting cancer cell proliferation.⁷

 

Similar to many cytotoxic anticancer drugs, 5-FU has a relatively narrow therapeutic window in which systemic administration to the drug causes both toxicity and efficacy to occur at the same time. The DPYD gene encodes DPD, an enzyme that  converts 5-FU into dihydro-5-FU (FUH2) and is responsible for 80%–90% of 5-FU clearance.⁷ According to the study, 5-FU is unstable in whole blood and plasma at room temperature, principally because DPD catabolizes 5FU by ex vivo catabolism.^{5, 8, 9, 10,11} A DPD inhibitor i.e., Gimeracil, added to the samples to stabilize 5-FU and obtain its maximum plasma concentration level.⁷

 

With this study we aim to develop a rapid and sensitive method using the UPLC  for measuring 5-fluorouracil(5-FU) levels in patients with gastrointestinal cancer. So that dossing strategy can improve therapeutic drug monitoring service to gain maximum therapeutic benefit with lesser toxicity.

 

MATERIALS AND METHODS:

Chemicals and materials:

A reference standard of 5-fluorouracil (Merck) was purchased (purity >99%). Reference standards of 5-bromouracil and Gimeracil (Sigma Aldrich) were purchased (purity >99%). Pure water was filtered by a Milli Q plus water purification system from Merck Millipore, MA, USA. HPLC grade solvents acetonitrile (ACN) and methanol (MeOH) were purchased (Merck). Perchloric acid was purchased. Human plasma was obtained after informed consent from the blood bank of the Advanced Center for Cancer Care, Research and Education, Navi Mumbai, India.

 

Stock solution, calibration and quality control samples:

The main stock solutions of 5FU and 5BU were prepared by dissolving 1 mg each in 1 mL (1000 µL) of diluent {methanol: water (1:1%v/v)} in microcentrifuge tubes. Stock solutions were stored in a refrigerator at -20°C. Different pooled working solutions of 5FU and its internal standard 5BU were prepared by diluting the stock solutions with 50% MeOH in water. Subsequently, 15μl of the pooled working solutions were diluted in 285μl of human plasma along with calibration standards were prepared at concentrations of 2 to 10ug/ml. Quality control (QC) samples were also prepared independently in blank plasma at four different concentrations lower limit of quantification [LLOQ], low QC, medium QC, and high QC) for 5FU with an internal standard of 5BU. All spiked samples were freshly prepared for each analysis.

 

Sample preparation:

An aliquot of 200µl of the calibration or QC standard or patient samples was prepared in 1.5ml microcentrifuge tubes, and 20µl of IS stock solution 5-bromouracil (10µg/ml) was added and vortexed in all samples except the blank sample. To these microcentrifuge tubes, 10µl of gimeracil (0.3mg/ml) was added and vortexed for 30seconds. 1000µl of 100% acetonitrile was then added and vortexed for 3minutes. The samples were then centrifuged at 13,000rpm for 13minutes. A total of 900µl of the supernatant was transferred to other microcentrifuge tubes and evaporated to dryness at 47°C under nitrogen using a Speedvac. The dried sample was reconstituted by adding 100µl of reconstitution solution containing 0.04% perchloric acid, vortexing for 3minutes and centrifuging at 13,000rpm for 13minutes. A total of 90µl of the supernatant was transferred to the labelled HPLC vials for analysis. For plasma blank, an aliquot of the plasma was prepared with an internal standard and processed further.

 

HPLC instrument and condition^12,13,14

This study involved the quantification of analytes through the use of a Dionex Ultra 3000 chromatography system manufactured by Thermo Fischer Scientific (Mao, United States). The system included a gradient pump, degasser, gradient mixer, UV diode array detector, autosampler, and chromatographic workstation (Chromeleon) manufactured by Dionex Corporation (California, United States). The chromatographic separation was performed in the mobile phase consisting of a mobile phase in the ratio of A: 100% methanol B: 0.04% perchloric acid in water. During the validation procedure, the chromatographic column of the study was kept at a temperature of 27^oC. Dual wavelength mode was used to detect the characteristic UV spectrum (range 200-800 nm) of 5FU and 5BU at 265nm and 280nm.

 

Method validation^{15,16,17,18,19,20,21}

Method validation was performed in accordance with FDA and ICH guidelines 2022.¹⁴

 

 

Limitations in quantification, detection, and resolution:

Signal-to-noise ratio calculations were used to determine the peak resolution of 5FU as well as the limit of detection (LOD) and LLOQ. Plasma samples spiked with 5FU and having a signal-to-noise ratio of at least 3:1 were used to measure LOD. The reduced LLOQ was evaluated at a signal-to-noise ratio of at least 10:1.

 

Selectivity:

Six separate plasma lots, one of which consisted of hemolyzed plasma, were used to evaluate selectivity. LLOQ and plasma blank were treated separately for each lot. The interference results were measured. A detector response in blank plasma, which was less than 20% LLOQ for the retention times of 5FU and 5BU, served as confirmation that no interfering components were present.

 

Sensitivity:

The sensitivity of 5FU with 5BU was established at the LLOQ level. Six LLOQ replicates were run. Accuracy and precision were observed for 5FU with 5BU. Sensitivity was defined as 80–120% precision and ± 20% accuracy.

 

Linearity range:

A 5FU calibration curve was constructed by regressing the fraction of the analyte peak area at IS on analyte concentrations in spiked plasma samples. Eight non-zero calibration benchmarks and quality measures were examined to coordinate the calibration curves. The confidence is considered to be just in the range where the precision of the concentrations calculated backwards from the slope of the condition is 20% of the nominal concentration of the LLOQ standard and 15% for all other guidelines.

 

Accuracy and precision:

By analysing QC samples (LLOQ, low, medium, and high QCs) in six replicates at three different concentrations along with calibration curve standards, precision and trueness were evaluated within one day and between two days. Each back-calculated concentration of QC standards was identified. For all QCs and for LLOQ, the acceptable range for accuracy and precision was 15% to 20%.

 

Ruggedness:

By estimating 5FU with 5BU using parameters like two individual analysts and different columns with similar specifications for the method created at LLOQ, low QC, medium QC, and high QC. For non-LLOQ and LLOQ samples, the acceptable limits for mean precision and accuracy were 15% and 20%, respectively.

 

Dilution integrity:

Dilution integrity was performed by diluting upper most concentration level i.e., ULOQ to its 1/2^th and 1/10^th dilution level. Five replicates of each dilution i.e., 1/2^th and 1/10^th respectively were injected on system with calibration curve standards and quality controls. QC. The acceptable limits for mean precision and accuracy were ±15% for non-LLOQ and ±20% for LLOQ samples.

 

Reinjection reproducibility:

Reinjection reproducibility was performed as long batch performance to check reproducibility of injections. Reinjection reproducibility was checked by injecting 10 replicates of each QCS-LLOQ, LQC, MQC, HQC with calibration curve standards batch. Results of reinjection reproducibility shows reproducibility of analyte and internal standard within acceptable limits for mean precision and accuracy were ±15% for non-LLOQ and ±20% for LLOQ samples.

 

Stability testing:

Stabilities tested during method validation are as follows:

 

Condition stabilities:

a)     Freeze thaw stability: Freeze thaw was performed on spiked LQC and HQC by following freeze and thaw cycle for 7 days and results was checked by processing fresh LQC and HQC six replicates with processing of spiked samples.

b)    Autosampler stability: This stability was checked by processing six replicates of LQC and HQC to store them in autosampler for 24hrs then integrating the results with six replicates of freshly prepared LQC and HQC.

c)     Dry extract stability: This stability was checked by processing six replicates of LQC and HQC and storing them without reconstitution for period then integrating the results with six replicates of freshly prepared LQC and HQC.

d)    Wet extract stability: This stability was checked by processing six replicates of LQC and HQC and storing them with reconstituting the processed samples for period then integrating the results with six replicates of freshly prepared LQC and HQC.

e)     Bench top stability: This stability was checked by spiking six replicates of LQC and HQC and storing them on bench for almost 6hrs then results was checked by processing fresh six replicates of LQC and HQC with analysis of spiked samples.

 

1.     Short term stock solution stability:

6 aliquots of LQC and HQC were spiked and kept in storage for 6hrs then after 6hrs 6 aliquots of freshly prepared LQC and HQC were processed along with stored QCs. Results were checked by comparing accuracy and precision.

2.     7^th day and 15^th day stock solution stability:

6 aliquots of LQC and HQC were spiked and kept in storage for 7^th and 15^th days, respectively then after 7^th and 15^th days 6 aliquots of freshly prepared LQC and HQC were processed along with stored QCs respectively on those days. Results were checked by comparing accuracy and precision.

 

The acceptable limits for mean precision and accuracy of all stabilities were ±15% for non-LLOQ and ±20% for LLOQ samples.

 

RESULTS:

System Suitability:

The %CV of the analyte area and IS area were found to be ≤ 0.275 and ≤ 2.193 respectively, for all analyte and IS on each day. The results are as follow:

 

Table 1: Results of System suitability

5 FU	Analyte area	IS area
PLS MQC 01	3.7183	0.613
PLS MQC 02	3.7239	0.6208
PLS MQC 03	3.7166	0.6348
PLS MQC 04	3.6998	0.6169
PLS MQC 05	3.7006	0.6058
PLS MQC 06	3.7051	0.5952
MEAN	3.711	0.614
SD	0.010	0.013
%RSD	0.275	2.193

 

Selectivity:

All six lots were found to be free of significant interferences at the retention time of analyte

 

Table 2: Results of selectivity

5 FU	Analyte area	IS area	% Difference for analyte	% Difference for IS
PLS BLK 01	0	0	0	0
LLOQ 01	0.2793	0.5901
PLS BLK 02	0	0	0	0
LLOQ 02	0.3788	0.5091
PLS BLK 03	0	0	0	0
LLOQ 03	0.2596	0.4999
PLS BLK 04	0	0	0	0
LLOQ 04	0.358	0.5213
PLS BLK 05	0	0	0	0
LLOQ 05	0.2601	0.4948
PLS BLK 06	0	0	0	0
LLOQ 06	0.2725	0.4934

 

Sensitivity:

The precision %CV for analyte and internal standard at LLOQ were observed as 0.418 and 3.757 % respectively which were within acceptance criteria.

 

Table 3: Results of Sensitivity

5 FU	Conc. (ug/ml)	Analyte area	IS area
LLOQ 01	0.2ug/mL	0.2271	0.4511
LLOQ 02		0.2294	0.4614
LLOQ 03		0.2285	0.4608
LLOQ 04		0.2274	0.4302
LLOQ 05		0.2291	0.4287
LLOQ 06		0.229	0.4258
	MEAN	0.228	0.443
	SD	0.001	0.017
	%RSD	0.418	3.757

 

Reinjection stability:

Table 4: Results of Reinjection stability

Levels (n=10)	Nominal Concentration (ug/ml)	%RSD	% Accuracy
LLOQ	0.20	9.22% to 14.51%	85.49% to 90.78%
LQC	0.31	0.95% to 4.17%	96.41% to 104.17%
MQC	2.5	0.35% to 13.06%	100.35% to 113.06%
HQC	7.5	9.75% to 14.14%	85.11% to 90.25%

 

Calibration Curve/Linearity:

The average correlation coefficient (R²) was observed ≥ 0.99 during experiment

 

Table 5: Results of linearity

Levels (ug/mL)	Mean	SD	% RSD	% Accuracy
0.2	0.3	0.0	8.8	99.71
0.31	0.4	0.0	8.0	99.55
0.62	0.9	0.1	8.7	99.11
1.25	1.7	0.2	10.4	98.27
2.5	3.4	0.1	4.3	96.63
5	6.8	0.5	7.8	93.20
7.5	10.5	0.7	7.0	89.54
10	13.5	0.8	5.7	86.50

 

Accuracy and precision:

Results of Accuracy was observed in between 89.28% to 99.74%.

 

Results of Precision (Intraday and Interday) %CV was observed in between 1.45% to 10.67%.

 

Table 6: Accuracy and precision (Intraday)

Levels	Nominal Concentration (ug/ml)	Calculated Concentration (ug/ml) SET 1	Calculated Concentration (ug/ml) SET 2
LLOQ	0.20	0.2661	0.2607
LQC	0.31	0.406	0.373
MQC	2.5	2.9827	3.2943
HQC	7.5	9.1474	9.6254

 

Tabel  7:% RSD and % Accuracy of Intraday

	Levels	Conc. (ug/ml)	Mean	SD	% RSD	% Accuracy
Intraday n=6	LLOQ	0.20	0.263	0.004	1.45	99.74
	LQC	0.31	0.390	0.023	5.99	99.61
	MQC	2.5	3.139	0.220	7.02	96.86
	HQC	7.5	9.386	0.338	3.60	90.61

 

Table 8: Accuracy and Precision (Interday)

Levels	Nominal Concentration (ug/ml)	Cal. Conc. (ug/ml) SET 1	Cal. Conc. (ug/ml) SET 2	Cal. Conc. (ug/ml) SET 3	Cal. Conc. (ug/ml) SET 4	Cal. Conc. (ug/ml) SET 5	Cal. Conc. (ug/ml) SET 6
LLOQ	0.20	0.2956	0.3021	0.2613	0.2713	0.3025	0.3020
LQC	0.31	0.4779	0.4779	0.3983	0.4375	0.4983	0.4745
MQC	2.5	3.6369	3.6369	2.9931	3.8943	3.5559	3.0109
HQC	7.5	10.6691	10.8321	9.1231	11.5326	10.9858	11.1775

 

Table 9: % RSD and % Accuracy of Interday

	Levels	Nominal Concentration (ug/ml)	Mean	SD	% RSD	% Accuracy
Interday n=6	LLOQ	0.20	0.289	0.018	6.28	99.71
	LQC	0.31	0.461	0.036	7.90	99.54
	MQC	2.5	3.455	0.369	10.67	96.55
	HQC	7.5	10.720	0.837	7.81	89.28

 

Autosampler carry over:

Table 10: Results of autosampler carryover

5FU	Conc. (ug/ml)	Analyte area	IS area	% Difference for analyte	% Difference for IS
PLS BLK	-	0	0	0.0	0
ULOQ	7.50	11.3223	0.5428
PLS BLK	-	0	0	0.0	0
LLOQ	0.20	0.2312	0.4837
AQ BLANK	-	0	0	0.0	0
AQ ULOQ	7.50	8.3786	0.3416
AQ BLANK	-	0	0	0.0	0
AQ LLOQ	0.20	0.1728	0.3562

 

Matrix effect:

Table 11: Results of matrix effect

 

Dilution integrity:

The accuracy of the ULLQ concentration dilution at 1/2 and 1/10 times is, respectively, 106.23% to 113.23% and 103.86% to 110.96%.

 

The %CV of dilutions of ULLQ by 1/2th and 1/10th, respectively, ranged from 8.88% to 13.24% and 3.86% to 10.93%.

 

 

Table 12: Results of dilution integrity (1/2^th times dilution)

Dilution integrity for (1/2th times dilution)
Nominal conc. (ug/ml) 10ug/ml	Diluted concentration upto	Calculated conc.(ug/ml)	% RSD	% Accuracy
	5ug/ml	5.66	13.24	113.24
		5.31	6.23	106.23
		5.42	8.33	108.33
		5.40	7.97	107.97
		5.44	8.88	108.88
		5.66	13.24	113.24

 

Table 13: Results of dilution integrity (1/10^th times dilution)

Dilution integrity for (1/10th times dilution)
Nominal conc. (ug/ml) 10ug/ml	Diluted concentration upto	Calculated conc.(ug/ml)	% RSD	% Accuracy
	1ug/ml	1.04	3.86	103.86
		1.11	10.93	110.93
		1.08	7.83	107.83
		1.06	5.88	105.88
		1.07	7.07	107.07
		1.04	3.86	103.86

 

Ruggedness:

Table 14: Results of ruggedness (Different column)

Ruggedness	Levels	Nominal concentration (ug/mL)	% RSD	% Accuracy
Different column	CS1	0.2	2.13	97.87
	CS2	0.31	2.33	102.33
	CS3	0.62	1.38	101.38
	CS4	1.25	1.10	98.90
	CS5	2.5	1.71	101.71
	CS6	5	0.91	99.09
	CS7	7.5	0.90	99.10
	CS8	10	0.44	100.44
	LQC	0.31	6.27	93.73
	MQC	2.5	3.47	103.47
	HQC	7.5	3.57	96.43

 

Table 15. Results of ruggedness (Different analyst)

Ruggedness	Levels	Nominal concentration (ug/mL)	% RSD	% Accuracy
Different analyst	CS1	0.2	17.39	117.39
	CS2	0.31	7.53	107.53
	CS3	0.62	3.91	103.91
	CS4	1.25	0.18	99.82
	CS5	2.5	0.47	99.53
	CS6	5	0.36	99.64
	CS7	7.5	3.24	96.76
	CS8	10	1.90	101.90
	LQC	0.31	10.84	110.84
	MQC	2.5	4.25	104.25
	HQC	7.5	3.19	103.19

 

Stabilities

Stock solution stabilities

i. Short term stock solution stability

ii. Long term stock solution stability

 

Table 16: Results of short-term stock solution stability

Short term stock solution stability (6hrs)		Mean	SD	% RSD
AQ MQC	Fresh	1.04	0.00	0.13
	After 6 hrs	1.05	0.00	0.18
PLS MQC	Fresh	3.28	0.01	0.27
	After 6 hrs	3.46	0.20	5.66

 

Table 17: Results of long-term stock solution stability

Long term stock solution stability			Mean	SD	% RSD
7th day stability	LQC	Fresh	0.48	0.00	0.77
		After 7th day	0.46	0.00	0.65
	HQC	Fresh	10.58	0.00	0.04
		After 7th day	9.38	0.01	0.08
15th day stability	LQC	Fresh	0.51	0.00	0.15
		After 15th day	0.47	0.00	0.53
	HQC	Fresh	11.49	0.01	0.10
		After 15th day	9.86	0.01	0.06
30th day stability	LQC	Fresh	0.48	0.00	0.16
		After 30th day	0.47	0.00	0.27
	HQC	Fresh	11.40	0.01	0.10
		After 30th day	11.11	0.01	0.13

 

Condition stabilities:

 

Table 18: Results of Condition stabilities

Freeze thaw stability
		Mean	SD	%RSD
LQC	Fresh	0.49	0.00	0.03
	After 7 cycles	0.51	0.00	0.24
HQC	Fresh	10.58	0.01	0.05
	After 7 cycles	9.67	0.02	0.22
Dry extract stability
LQC	Fresh	0.49	0.00	0.03
	Extract stability after 6hrs	0.50	0.00	0.41
HQC	Fresh	10.58	0.01	0.05
	Extract stability after 6hrs	10.44	0.01	0.09
Wet extract stability
LQC	Fresh	0.49	0.00	0.03
	Extract stability after 6hrs	0.51	0.00	0.41
HQC	Fresh	10.58	0.01	0.05
	Extract stability after 6hrs	10.71	0.01	0.14

 

Auto-sampler stability
LQC	Fresh	0.49	0.00	0.03
	After 12 hrs	0.47	0.00	0.92
HQC	Fresh	10.58	0.01	0.05
	After 12 hrs	10.76	0.01	0.08
Benchtop stability
LQC	Fresh	0.49	0.00	0.03
	After 6 hrs	0.45	0.00	0.20
HQC	Fresh	10.58	0.01	0.05
	After 6 hrs	10.76	0.01	0.12

 

CONCLUSION:

The method was successfully developed and validated to measure the amount of 5FU present in plasma samples of cancer patients. This method was used as bioanalysis to observe the therapeutic drug monitoring of patients with GI cancer.

 

ACKNOWLEDGMENT:

The authors are thankful to Management of Gahlot Institute of Pharmacy, Koparkhairane, for their support during this study. The authors are grateful to Dr. Vikran Gota and team of Advanced Center for Cancer Treatment, Research and Education, Kharghar, Navi Mumbai (ACTREC) for providing the necessary facilities to carry out the work.

 

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Accepted on 25.02.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(5):2345-2351.