Determination of Capecitabine and Its Metabolites in Plasma of Egyptian Colorectal Cancer Patients
Abstract
:1. Introduction
2. Materials and Methods
2.1. HPLC Method Development for the Determination of Capecitabine and Its Metabolites Using HPLC-UV
2.1.1. Chemicals and Reagents
2.1.2. Equipment and Software
2.1.3. The Choice of Column and Method Development Strategy
Run | Column | Tg (Min) | T (°C) | Mobile Phase A | Mobile Phase B | %B Range | Detection | Flow Rate | Injection Volume |
---|---|---|---|---|---|---|---|---|---|
RUN 1 (Figure 3C) | Hypersil Gold C18 | 20 | 25 | Acidified water with phosphoric acid, pH 2.6 | Methanol | 5–100% B | 205, 266, 310 (nm) | 1 (mL/min) | 25 (µL) |
RUN 2 (Figure 3D) | Hypersil Gold C8 | 20 | 25 | ||||||
RUN 3 (Figure 3E) | 20 | 25 | 20 mM ammonium acetate at pH 4.0 | ||||||
RUN 4 (Figure 3F) | 20 | 25 | 5.0 mM tetrabutylammonium hydrogen sulfate at pH 8.0 | ||||||
DRYLAB1 (Figure 4A) | synergi fusion RP | 20 | 25 | Acidified water with phosphoric acid, pH 2.6 | |||||
DRYLAB2 (Figure 4B) | 60 | 25 | |||||||
DRYLAB3 (Figure 4C) | 60 | 45 | |||||||
DRYLAB4 (Figure 4D) | 20 | 45 | |||||||
OPTIMUM (Figure 4E) | 27 | 45 | 0–7 min (5–75% B), 7–20 min (75–100% B) |
2.2. Validation of the Newly Developed HPLC Method
2.3. Analytes Extraction from Plasma and Assay Application to Patient Plasma Samples
3. Results
3.1. Analytical Method Development
3.1.1. Preliminary Trials for the Choice of a Suitable Analytical Column
3.1.2. Computer-Assisted Analytical Method Optimization
3.2. Validation of the Developed HPLC Method
3.3. A Prospective Study on Egyptian Colorectal Cancer Patients
3.4. Correlations between Plasma Concentration of FU, DFCR, DFUR and Toxicity
4. Discussion
5. Conclusions
6. Recommendations
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CCB | Capecitabine |
CDD | cytidine deaminase |
CLU | 5-Chlorouracil |
DFCR | 5′-deoxy-5-fluorocytidine |
CES | carboxylesterase |
DFUR | 5′-deoxy-5-fluorouridine |
DHFU | 5,6-dihydro-5-fluorouracil |
DPD | Dihydropyrimidine Dehydrogenase |
HPLC | high-pressure liquid chromatography |
FBAL | α-fluoro-β-alanine |
FU | 5-Fluorouracil |
FUPA | 5-fluoro-ureidopropionic acid |
IS | internal standard |
LC | liquid chromatography |
SNPs | single nucleotide polymorphisms |
TP | thymidine phosphorylase |
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Column Type | Source | Particle Size | Dimensions (Mm) | Features |
---|---|---|---|---|
HYPERSIL GOLD C18 | Thermo Scientific, Waltham, MA, USA | 3 μm | 150 × 4.6 | C18 (Commonly used in pharmaceutical and biomedical applications) |
HYPERSIL GOLD C8 | Thermo Scientific | 5 μm | 250 × 4.6 | C8 |
SYNERGI FUSION-RP | Phenomenex, Torrance, CA, USA | 4 μm | 150 × 4.6 | Polar embedded ligand and hydrophobic ligand, Tetramethylsilane (TMS) end capping. Stable within a pH range of 1.5–10.0 [15] |
Analyte | DHFU | FU | DFCR | DFUR | CCB |
---|---|---|---|---|---|
Conc 1 (ng/mL) | 10 | 1 | 1 | 1 | 10 |
Conc 2 (ng/mL) | 1 ∗ 102 | 1 ∗ 102 | 1 ∗ 102 | 1 ∗ 102 | 1 ∗ 102 |
Conc 3 (ng/mL) | 1 ∗ 103 | 1 ∗ 103 | 1 ∗ 103 | 1 ∗ 103 | 1 ∗ 103 |
Conc 4 (ng/mL) | 1 ∗ 104 | 1 ∗ 104 | 1 ∗ 104 | 1 ∗ 104 | 1 ∗ 104 |
Conc 5 (ng/mL) | 2 ∗ 104 | 2 ∗ 104 | 1 ∗ 105 | 2 ∗ 104 | 2 ∗ 104 |
Parameter | DHFU | FU | DFCR | DFUR | CCB |
---|---|---|---|---|---|
LINEARITY RANGE (NG/ML) | From 10 to 2 × 104 | From 1 to 2 × 104 | From 1 to 1 × 105 | From 1 to 2 × 104 | From 10 to 2 × 104 |
CORRELATION COEFFICIENT (R) | >0.98 | >0.98 | >0.98 | >0.98 | >0.98 |
INTRA-DAY PRECISION (CV%) | <15% | <15% | <15% | <15% | <15% |
INTER-DAY PRECISION (CV%) | <10% | <10% | <10% | <10% | <10% |
PRE-STUDY CALIBRATION CURVE CV% | 1.1–4.9% | 1.6–7.9% | 1.2–4.7% | 1.7–6.7% | 1.6–8.4% |
ROUTINE QC SAMPLE CV% | 2.79–5.36% | 2.69–9.14% | 1.5–10.6% | 2.69–9.14% | 1.5–10.6% |
PRE-STUDY CALIBRATION CURVE %BIAS | −4.7% to 5.5% | −3.6% to 6.8% | 1.5% to 6.6% | 1.2% to 3.2% | −3.8% to 3.5% |
ROUTINE QC SAMPLE %BIAS | −7.2% to 8.9% | 1.7% to 4.2% | −1.27% to 5.7% | 1.7% to 4.2% | −1.27% to 5.7% |
LOD (NG/ML) | 3 ng/mL | 0.3 ng/mL | 0.3 ng/mL | 0.3 ng/mL | 3 ng/mL |
LOQ (NG/ML) | 10 ng/mL | 1 ng/mL | 1 ng/mL | 1 ng/mL | 10 ng/mL |
P# | Sex | Age | Regimen | Conc. of FU (×103 ng/mL) | Conc. of DFCR (×103 ng/mL) | Conc. of DFUR (×103 ng/mL) | Toxicity Grade | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
D1 C1 | D14 C1 | D1 C2 | D1 C1 | D14 C1 | D1 C2 | D1 C1 | D14 C1 | D1 C2 | A | N | V | D | C | F | M | H | ||||
1 | M | 46 | CCB | 43 | 37 | 33 | 2 | 6 | 0 | 4 | 11 | 1 | 0 | 1 | 0 | 2 | 1 | 0 | 1 | 3 |
2 | M | 44 | CAPOX | 55 | 52 | 31 | 14 | 1 | 2 | 7 | 1 | 1 | 1 | 1 | 0 | 3 | 1 | 1 | 1 | 2 |
3 | M | 63 | CAPOX | 58 | 98 | 0 | 3 | 1 | 0 | 3 | 2 | 0 | 2 | 1 | 2 | 1 | 0 | 0 | 0 | 1 |
4 | F | 55 | CAPOX | 44 | 26 | 0 | 0 | 2 | 0 | 13 | 8 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 |
5 | F | 60 | CCB | 122 | 50 | 41 | 1 | 2 | 514 | 3 | 2 | 1 | 1 | 1 | 0 | 1 | 0 | 0 | 0 | 0 |
6 | M | 31 | CAPOX | 70 | 85 | 132 | 2 | 4 | 0 | 1 | 3 | 2 | 0 | 1 | 1 | 0 | 1 | 1 | 0 | 2 |
7 | M | 33 | CCB | 50 | 80 | 0 | 12 | 5 | 0 | 4 | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
8 | F | 36 | CAPOX | 52 | 0 | 70 | 5 | 0 | 6 | 10 | 0 | 3 | 1 | 0 | 2 | 0 | 0 | 0 | 0 | 0 |
9 | F | 51 | CAPOX | 28 | 81 | 37 | 1 | 698 | 1 | 1 | 3 | 2 | 1 | 1 | 2 | 2 | 2 | 0 | 0 | 1 |
10 | M | 70 | CAPOX | 50 | 77 | 62 | 8 | 1 | 6 | 5 | 1 | 3 | 2 | 0 | 0 | 0 | 3 | 0 | 2 | 0 |
11 | M | 32 | CAPOX | 24 | 27 | 28 | 0 | 2 | 3 | 2 | 2 | 3 | 3 | 0 | 3 | 2 | 2 | 0 | 1 | 0 |
12 | M | 64 | CCB | 78 | 47 | 0 | 21 | 0 | 0 | 3 | 1 | 0 | 0 | 1 | 2 | 0 | 1 | 1 | 1 | 3 |
13 | F | 62 | CAPOX | 102 | 48 | 31 | 2 | 2 | 0 | 2 | 3 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 0 | 3 |
14 | F | 47 | CAPOX | 35 | 38 | 18 | 1 | 2 | 1 | 3 | 3 | 4 | 2 | 2 | 2 | 3 | 1 | 1 | 2 | 3 |
15 | M | 37 | CAPOX | 28 | 39 | 45 | 3 | 1 | 1 | 5 | 2 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
16 | M | 48 | CAPOX | 31 | 86 | 146 | 5 | 9 | 1 | 4 | 8 | 24 | 0 | 1 | 0 | 1 | 2 | 0 | 1 | 3 |
17 | M | 58 | CAPOX | 13 | 16 | 18 | 5 | 1 | 1 | 5 | 1 | 1 | 2 | 1 | 0 | 3 | 1 | 1 | 1 | 0 |
18 | M | 73 | CAPOX | 54 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 0 |
19 | F | 54 | CAPOX | 40 | 49 | 29 | 2 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 0 | 0 |
20 | F | 68 | CAPOX | 58 | 0 | 0 | 1 | 0 | 0 | 6 | 0 | 0 | 2 | 1 | 1 | 0 | 1 | 0 | 0 | 3 |
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Shamseldin, S.; Botros, L.S.; Salem, S.E.; Abdel-Maksoud, S.; Gad, M.Z.; Hanafi, R.S. Determination of Capecitabine and Its Metabolites in Plasma of Egyptian Colorectal Cancer Patients. Analytica 2023, 4, 397-414. https://doi.org/10.3390/analytica4040029
Shamseldin S, Botros LS, Salem SE, Abdel-Maksoud S, Gad MZ, Hanafi RS. Determination of Capecitabine and Its Metabolites in Plasma of Egyptian Colorectal Cancer Patients. Analytica. 2023; 4(4):397-414. https://doi.org/10.3390/analytica4040029
Chicago/Turabian StyleShamseldin, Sara, Liza Samir Botros, Salem Eid Salem, Sahar Abdel-Maksoud, Mohamed Zakaria Gad, and Rasha Sayed Hanafi. 2023. "Determination of Capecitabine and Its Metabolites in Plasma of Egyptian Colorectal Cancer Patients" Analytica 4, no. 4: 397-414. https://doi.org/10.3390/analytica4040029