Sensitive and Cost-Effective TLC-Densitometric Method for Determination of Metronidazole and Tinidazole in Tablets
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reference Standards
2.2. Standard Solutions of Active Pharmaceutical Ingredients (APIs)
2.3. Solutions of Metronidazole and Tinidazolum
2.4. TLC-Densitometry
2.5. TLC Method Validation
2.6. Quantitative Analysis of Metronidazole and Tinidazole in Tablets and Comparison with Pharmacopeial Method
2.7. Statistical Analysis
3. Results and Discussion
3.1. Validation
3.1.1. Optimization of Chromatographic Conditions
3.1.2. Linearity and Range
3.1.3. Precision
3.1.4. Accuracy
3.1.5. Limit of Detection (LOD) and Limit of Quantification (LOQ)
3.1.6. Robustness
3.2. Quantitative Analysis of M and T in Tablets and Comparison with Pharmacopeial Method
4. Conclusions
Supplementary Materials
Funding
Data Availability Statement
Conflicts of Interest
References
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No of Mobile Phase a | Average RF and Rs Values | |
---|---|---|
2 | RF | RF(M) = 0.35 ± 0.02, RF(O) = 0.43 ± 0.02, RF(T) = 0.50 ± 0.02, RF(S) = 0.57 ± 0.02, RF(IMP) = 0.64 ± 0.03; |
RS | RS(M/O) = 0.78, RS(O/T) = 0.77, RS(T/S) = 0.83, RS(S/IMP) = 0.80 | |
8 | RF | RF(IMP) = 0.36 ± 0.02, RF(M) = 0.41 ± 0.02, RF(O) = 0.46 ± 0.03, RF(S) = 0.51 ± 0.03, RF(T) = 0.59 ± 0.03; |
RS | RS(IMP/M) = 0.75, RS(M/O) = 1.07, RS(O/S) = 1.05, RS(S/T) = 1.29 | |
9 | RF | RF(IMP) = 0.31 ± 0.02, RF(M) = 0.34 ± 0.02, RF(S) = 0.39 ± 0.02, RF(O) = 0.47 ± 0.03, RF(T) = 0.59 ± 0.03; |
RS | RS(IMP/M) = 0.83, RS(M/S) = 1.27, RS(S/O) = 1.43, RS(O/T) = 1.78 | |
12 | RF | RF(M) = 0.45 ± 0.02, RF(IMP) = 0.49 ± 0.02, RF(O) = 0.55 ± 0.03, RF(S) = 0.65 ± 0.02, RF(T) = 0.74 ± 0.03; |
RS | RS(M/IMP) = 0.83, RS(IMP/O) = 1.33, RS(O/S) = 1.33, RS(S/T) = 1.33 | |
13 | RF | RF(M) = 0.39 ± 0.02, RF(IMP) = 0.42 ± 0.02, RF(O) = 0.50 ± 0.03, RF(S) = 0.61 ± 0.02, RF(T) = 0.67 ± 0.02; |
RS | RS(M/IMP) = 0.53, RS(IMP/O) = 1.18, RS(O/S) = 1.58, RS(S/T) = 1.13 | |
14 | RF | RF(M) = 0.28 ± 0.02, RF(S) = 0.35 ± 0.02, RF(T) = 0.41 ± 0.03, RF(O) = 0.47 ± 0.03, RF(IMP) = 0.56 ± 0.03; |
RS | RS(M/S) = 0.88, RS(S/T) = 1.07, RS(T/O) = 1.07, RS(O/IMP) = 1.33 | |
16 | RF | RF(M) = 0.35 ± 0.02, RF(S) = 0.41 ± 0.02, RF(T) = 0.51 ± 0.02, RF(O) = 0.54 ± 0.02, RF(IMP) = 0.63 ± 0.03 |
RS | RS(M/S) = 1.05, RS(S/T) = 1.44, RS(T/O) = 0.30, RS(O/IMP) = 1.20 | |
19 | RF | RF(IMP) = 0.30 ± 0.02, RF(M) = 0.38 ± 0.03, RF(S) = 0.44 ± 0.03, RF(O) = 0.51 ± 0.03, RF(T) = 0.70 ± 0.04 |
RS | RS(IMP/M) = 1.33, RS(M/S) = 1.22, RS(S/O) = 1.29, RS(O/T) = 2.10 |
Method Characteristic | 5-Nitroimidazole | ||
---|---|---|---|
Metronidazole | Tinidazole | ||
Retardation factor (Rf) | 0.38 ± 0.03 | 0.70 ± 0.04 | |
Range ([μg/spot)] | 0.2–2.0 | 0.2–2.0 | |
Linearity ([μg/spot)] A = a·X + b | a | 7340.4 (±133.1) | 7828.0 (±114.2) |
b | 3071.5 (±140.2) | 5567.9 (±130.1) | |
n | 10 | 10 | |
r | 0.9989 | 0.9992 | |
s | 235.6 | 218.6 | |
F | 3557 | 4701 | |
p | <0.0001 | <0.0001 | |
LOD ([µg/spot)] SD | 0.012 0.001 | 0.022 0.001 | |
LOQ ([µg/spot)] SD | 0.036 0.003 | 0.066 0.003 | |
For tablets | |||
Accuracy (n = 6) | |||
for 50% standard added | R = 103.8%; CV = 1.96% | R = 101.8%; CV = 0.95% | |
for 100% standard added | R = 104.3%; CV = 1.13% | R = 99.1%; CV = 2.31% | |
for 150% standard added | R = 101.2%; CV = 2.48% | R = 100.9%; CV = 2.49% | |
Average recovery | 103.1% | 100.6% | |
Precision (CV, ([%])) | |||
Intraday (n = 3) | |||
for 1.75 µg/spot | 1.08 | 0.76 | |
for 1.00 µg/spot | 1.12 | 0.89 | |
for 0.30 µg/spot | 0.99 | 1.28 | |
Interday (n = 3) | |||
for 1.75 µg/spot | 1.33 | 0.99 | |
for 1.00 µg/spot | 1.39 | 1.44 | |
for 0.30 µg/spot | 1.48 | 1.76 | |
Robustness (CV, ([%])) | robust | robust |
Method | Mobile Phase | LOD and LOQ ([µg/spot)] | Ref. |
---|---|---|---|
Metronidazole | |||
HPTLC | MeOH + TCM (9:1, v/v) | LOD = 0.61 LOQ = 0.95 | [18] |
HPTLC | TLN + EA + MeOH + AM (3:1.5:0.5:0.1, v/v) | LOD = 0.046 LOQ = 0.116 | [19] |
HPTLC | Bz + EA + TLN + MeOH + GAcOH (9.5:2:5:1.5:0.5, v/v) | LOD = 0.88 LOQ = 1.93 | [21] |
TLC | ACN + AM + MeOH + MCL+ Hx (1.3:1.1:2:3:1, v/v) | LOD = 0.32 LOQ = 0.96 | [22] |
TLC | EA + Ace + Hx + AM (9.5:0.5:0.3:0.3, v/v) | LOD = 0.13 LOQ = 0.38 | [23] |
TLC | TCM + Ace + GAcOH (7.5:2.5:0.1, v/v) | LOD = 0.51 LOQ = 1.55 | [16] |
TLC | TCM + MeOH (9:1, v/v) | LOD = 0.052 LOQ = 0.159 | [36] |
Tinidazole | |||
HPTLC | TLN + EA + MeOH + -TEA (5.5:1.0:1.0:0.1, v/v) | LOD = 0.011 LOQ = 0.037 | [25] |
HPTLC | Ace + EtOH + 2% watery sodium dodecyl sulfate (WSDS) (3:4:2, v/v) | LOD = 0.0067 LOQ = 0.0203 | [33] |
HPTLC | 30% Trifluoroacetic acid (TFAA) | LOD a = 0.01 LOQ a = 0.03 LOD b = 0.12 LOQ b = 0.36 | [32] |
TLC | IP + butanol (BT) + AM + water (W) (25:50:5:25, v/v) | LOD = 0.1 LOQ = 0.3 | [30] |
TLC | MCL + IP + ACN + AM (11:1.2:5:0.2, v/v) | LOD = 0.2 LOQ = 0.6 | [27] |
TLC | TCM + MeOH (9:1, v/v) | LOD = 0.058 LOQ = 0.174 | [36] |
Experiment No | X1 | X2 | X3 | X4 | X5 | X6 | X7 | Content of Active Pharmaceutical Ingredient (yi) ([mg·tablet−1]) | ||
---|---|---|---|---|---|---|---|---|---|---|
M | T | |||||||||
1 | + | + | + | + | + | + | + | 493.8 | 494.2 | |
2 | + | + | − | + | − | − | − | 489.9 | 502.5 | |
3 | + | − | + | − | − | + | − | 496.9 | 495.2 | |
4 | + | − | − | − | + | − | + | 500.2 | 506.1 | |
5 | − | + | + | − | + | − | − | 502.8 | 499.5 | |
6 | − | + | − | − | − | + | + | 493.1 | 490.2 | |
7 | − | − | + | + | − | − | + | 493.2 | 493.5 | |
8 | − | − | − | + | + | + | − | 513.8 | 508.2 | |
Size of effect | M | −5.525 | −6.125 | −2.575 | −0.575 | 9.375 | 2.875 | −5.775 | ||
T | 1.650 | −4.150 | −6.150 | 1.850 | 6.650 | −3.450 | −5.350 | |||
The label claim ([mg)] | 500 | 500 | ||||||||
Average amount ([mg]) | 498.0 | 498.7 | ||||||||
Variance | 58.4 | 41.7 | ||||||||
Standard deviation (SD) | 7.64 | 6.46 | ||||||||
Coefficient of variation (CV, %) | 1.5 | 1.3 |
Metronidazole | Tinidazole | |||
---|---|---|---|---|
Method | ||||
A | B | A | B | |
Number of analyses | 10 | 10 | 10 | 10 |
1 | 510.3 | 528.6 | 489.9 | 492.5 |
2 | 523.0 | 506.4 | 495.2 | 488.9 |
3 | 520.0 | 521.5 | 492.3 | 512.6 |
4 | 495.6 | 516.2 | 487.6 | 501.8 |
5 | 503.6 | 508.3 | 502.6 | 481.7 |
6 | 488.4 | 498.2 | 509.9 | 479.6 |
7 | 520.1 | 501.3 | 487.1 | 509.9 |
8 | 515.3 | 496.3 | 512.3 | 508.1 |
9 | 491.7 | 517.8 | 507.8 | 505.5 |
10 | 496.7 | 497.5 | 508.8 | 491.8 |
Average | 506.5 | 509.2 | 499.4 | 497.2 |
Label claimed | 500 | 500 | 500 | 500 |
Amount of metronidazole and tinidazole (%) in relation to the label claim | 101.3 | 101.8 | 99.8 | 99.4 |
Standard deviation (SD) | 12.9 | 11.3 | 10.0 | 11.9 |
Coefficient of variation (CV, %) | 2.55 | 2.22 | 2.00 | 2.39 |
Confidence interval of arithmetic mean with confidence level equal to 95% | µ = 506.5 ± 9.2 | µ = 509.2 ± 8.1 | µ = 499.4 ± 7.2 | µ = 497.2 ± 8.5 |
t calculated | 0.497 | 0.447 | ||
t(95%·18) tabulated | 2.101 | 2.101 | ||
F calculated | 1.30 | 1.42 | ||
F(95%·f1 = f2 = 9) tabulated | 3.18 | 3.18 |
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Pyka-Pająk, A. Sensitive and Cost-Effective TLC-Densitometric Method for Determination of Metronidazole and Tinidazole in Tablets. Processes 2024, 12, 643. https://doi.org/10.3390/pr12040643
Pyka-Pająk A. Sensitive and Cost-Effective TLC-Densitometric Method for Determination of Metronidazole and Tinidazole in Tablets. Processes. 2024; 12(4):643. https://doi.org/10.3390/pr12040643
Chicago/Turabian StylePyka-Pająk, Alina. 2024. "Sensitive and Cost-Effective TLC-Densitometric Method for Determination of Metronidazole and Tinidazole in Tablets" Processes 12, no. 4: 643. https://doi.org/10.3390/pr12040643