Bioanalytical validation could be performed according to EMA or FDA Guidance. Both documents have similarities but are not identical. For example, the acceptance criteria for accuracy have been reported in EMA Guidance to the mean concentrations, but it is unclear if the FDA’s criteria are related to the mean or to each sample. Only the EMA specifies how to study matrix effect not only recommendations like FDA. EMA Guidance shows in more precise way the practical conduct of experiments, while the FDA shows reporting recommendations in more comprehensive way. Among the benefits of using EMA guidance are that found to have more flexibility regarding whether the information established in set in SOPs or study plans and supply the ability to laboratories having GLP certification to construct quality system fitting their own purpose. The EMA supply a consistent presentation of numerical values through using accurate formulas such as calculating accuracy and precision.1
Generally, using biological specimens in performing laboratory drug testing can provide various levels of sensitivity, specificity accuracy. Urine is mostly preferred biological specimen as it is easy collected also high concentrations of drug & metabolites can be found in urine this permits longer detection times than concentrations in serum13 .
In vitro tests are performed in the laboratory, and it involves the study of microorganisms, human or animal cells in culture. This methodology helps in evaluation different biological phenomena in certain cells without the distractions and potential confounding variables present in whole organisms.
Researchers can conduct more detailed analyses and investigate biological effects in a larger number of in vitro subjects than they would in animal or human trials. Despite positive preclinical findings, approximately 30% of drug candidates fail human clinical trials because of unwanted effects. An additional 60% do not achieve the desired effect.14
PRU is extensively eliminated unchanged in urine 60–65%. By taking in consideration the benefit of PRU native fluorescence besides the easiness of spectrofluorimetric procedure, a spectrofluorimetric method was validated to determine PRU in urine of human using small amount of urine (30µL) and deionized water that was chosen for its best sensitivity beside its greenness character.
3.1. Method optimization:
The results of studying the effectiveness of various diluting solvents were recorded. Figure 2 illustrate that the deionized water provides the greatest FI value. The selection of wavelengths of both excitation & emission wavelengths was tested depending on the ability of the used instrument to scan PRU excitation & emission wavelengths. PRU has 3 excitation wave lengths at 220, 272& 310nm. The later was chosen as best excitation wavelength although its low sensitivity because it gives least blank reading, Fig. 3 & Fig. 4. While 362 nm was found to be the optimal emission wavelength in terms of sensitivity as shown in Fig. 5.
3.2. Validation procedures:
Bioanalysis concerned with identifying and quantifying of analytes in different biological matrices. Validation aids in achievement of dependable results which are demanded for proper drug dosing and keep patient safety. Validation of this designed analytical procedure obeys the EMA Guidelines for Bioanalytical Method Validation 12.
3.2.1. Selectivity:
Six individual sources of blanks of urine matrix that individually analyzed to exclude any interfering components excreted with PRU and there is no interference is recorded.
3.2.2. Plotting calibration curve:
Six calibration curves have been constructed using various concentrations ranging from0.75 to 5.5 µg/mL PRU providing linear relationship and excellent recoveries (Table 1).
Table 1
Data obtained from the different calibration curves
Calibration number
|
Slope
|
Intercept
|
r
|
1
|
113.47
|
101.64
|
0.9996
|
2
|
112.89
|
97.128
|
0.9996
|
3
|
111.56
|
85.705
|
0.9993
|
4
|
96.28
|
106.85
|
0.9995
|
5
|
111.29
|
91.121
|
0.9999
|
6
|
110.33
|
98
|
0.9995
|
*Mean of slope = 109.30 |
*Mean of intercept = 96.74 |
*7 points were used in each calibration curve |
3.2.3. Lower limit of quantification (LLOQ):
The analyte signal of the LLOQ sample should be at least 5 times the signal of a blank sample as shown in Fig. 3 & Fig. 4.
The lower limit of quantification (LLOQ) is found to be 0.75 µg/mL. The %CV was calculated. And found to be 1.49, and it is considered an accepted value.
3.2.4 Accuracy & precision:
Within and between run accuracy and precision for PRU were estimated by using QC samples as illustrated in (Table 2) giving accepted values.
Table 2
Results of accuracy and precision for determination of PRU in human urine
|
Within-run
|
Between-run
|
LLOQ
|
99.36 ± 7.38
|
99.85 ± 9.87
|
QCL
|
104.21 ± 7.27
|
102.13 ± 9.00
|
QCM
|
99.73 ± 1.99
|
101.17 ± 4.73
|
QCH
|
100.44 ± 2.51
|
98.84 ± 5.33
|
n
|
5
|
5
|
3.2.5. Recovery:
This technique was carried out directly without extraction thus recovery is supposed to be around 100%. The presence of urine suppressed the FI, results were consistent in all the QC samples. Although the matrix effect of urine was high in suppressing PRU native fluorescence (around 15%) but the FI is still high to give the required sensitivity (Table 3).
Table 3: Matrix effect of PRU.
3.2.6. Evaluation of stability:
Short-term stability: FI of three determinations were recorded using low and high QC samples that were kept in room temperature for 24 hours
Freeze and thaw stability: The stability of PRU after three freeze- thaw cycles was determined by recording FI of three determinations using low & high QC samples that were exposed to three freeze–thaw cycles of − 80 ° C in three consecutive days
Long term stability: it was studied where PRU in urine matrix stored in freezer at − 80°C for 30 days using low & high QC samples then recording FI of three determinations.
Generally, samples are considered be stable as the results are within of the original concentration ± 15% of the nominal concentration. All stability samples compared against a calibration curve of freshly spiked calibration standards. The results discussed briefly in (Table 4).
Table 4
|
Mean recovery ± RSD % (n = 3)
|
|
short term stability
|
Freeze thaw cycle
|
Long term stability
|
QCL
|
103.51 ± 5.79
|
94.77 ± 3.59
|
92.65. ± 6.53
|
QCH
|
99.58 ± 1.78
|
98.21 ± 3.26
|
95.28 ± 3.70
|
3.2.7 Analysis of PRU in Dosage Form & evaluation of standard addition technique:
The proposed method was applied for the analysis of PRU dosage from after extraction and spiking in human urine and good recoveries were obtained as shown in Table 5. Also to assess the validity of the method on dosage form, application of standard addition technique was performed using 1, 1.25, 2, 2.5 µg of standard PRU, where good recoveries were obtained as shown in Table 6.
Table 5
Application of the proposed method for the determination of PRU in a pharmaceutical dosage form and application of the standard addition technique.
Preparation
|
Drug/Claimed potency
|
Claimed taken
|
%Found
|
Resolor® tablets
B.N. JGL5A00
|
PRU/ 2 mg per tablet
|
2µg/mL
|
94.65 ± 1.47
|
*Average of three determinations |
Table 6
Application of the standard addition technique.
Standard Addition Technique
|
Pure Added (µg/mL)
|
%Recovery (Mean ± SD)
|
1
|
100.41 ± 0.30
|
1.25
|
100.56 ± 1.00
|
2
|
98.48 ± 0.58
|
2.5
|
98.11 ± 0.83
|
*Average of three determinations |
3.3. Greenness evaluation of the recommended spectrofluorimetric method:
Analytical GAPI approach 15 is utilized to evaluate& quantify various environmental factors involved in steps of analytical methodology. Plotka-Wasylka presented the green analytical procedure index (GAPI) which acts as a combination of benefits of both the NEMI and Eco-scale. Also, it can evaluate the green character in the suggested method. GAPI can assess any analytical procedure using 15 factors, beginning with the sample preparation, reagents and solvents, instrumentation, waste and waste treatment. As shown in Table 7, all the assessed parameters have been represented in a colorful picogram based on their environmental impact that is either green, yellow or red according to the table listed in the GAPI approach Fig. 6.
Table 7
Greenness assessment using GAPI approach.
Category
|
Green
|
Yellow
|
Red
|
Sample preparation
|
Collection (1)
|
In-line
|
On-line or at-line
|
Off-line
|
Preservation (2)
|
None
|
Chemical or physical
|
Physico-chemical
|
Transport (3)
|
None
|
Required
|
-
|
Storage (4)
|
None
|
Under normal conditions
|
Under special conditions
|
Type of method: direct or indirect (5)
|
No sample preparation
|
Simple procedures, eg. filtration, decantation
|
Extraction required
|
Scale of extraction
(6)
|
Nano-extraction
|
Micro-extraction
|
Macro-extraction
|
Solvents/reagents used (7)
|
Solvent-free methods
|
Green solvents/reagents used
|
Non-green solvents/reagents used
|
Additional treatments (8)
|
None
|
Simple treatments (clean up, solvent removal, etc.)
|
Advanced treatments
(Derivatization, mineralization, etc.)
|
Reagent and solvents
|
Amount (9)
|
< 10 mL (< 10 g)
|
10–100 mL (10–100 g)
|
> 100 mL (> 100 g)
|
Health hazard (10)
|
Slightly toxic, slight irritant; NFPA health hazard score = 0 or 1.
|
Moderately toxic; could cause temporary incapacitation; NFPA = 2 or
3.
|
Serious injury on short-term exposure; known or
suspected small animal carcinogen; NFPA = 4.
|
Safety hazard (11)
|
Highest NFPA flammability or
instability score of 0 or 1. No special hazards.
|
Highest NFPA flammability or instability score of 2 or 3, or a special hazard is used.
|
Highest NFPA flammability or instability score of 4.
|
Instrumentation
|
Energy (12)
|
≤ 0.1 kWh per sample
|
≤ 1.5 kWh per sample
|
> 1.5 kWh per sample
|
Occupational hazard
(13)
|
Hermetic sealing of analytical process
|
-
|
Emission of vapours to the atmosphere
|
Waste (14)
|
< 1 mL (< 1 g)
|
1–10 mL (1–10 g)
|
> 10 mL (< 10 g)
|
Waste treatment (15)
|
Recycling
|
Degradation, passivation
|
No treatment
|
ADITIONAL MARK: QUANTIFICATION
|
Circle in the middle of GAPI: Procedure for qualification and quantification
|
No circle in the middle of GAPI: Procedure only for qualification
|
NFPA: National Fire Protection Association
|