AAS was qualified as per pharmacopoeia requirements. Copper, manganese, mercury and cadmium hallow cathode lamps were used for qualification purposes. Design qualification and installation qualification met the criteria of user requirements. Wavelength accuracy and reproducibility, resolution and baseline stability are in compliance with user requirements. Characteristics concentration or sensitivity, detection limit and characteristics mass were performed as a part of operational qualification which is 0.062 μg/mL, 0.00354 μg/mL, 1.41 pg respectively. Results of % RSD for precision study is 0.352 and 0.487 for flame and graphite furnace mode respectively fulfilled criteria of qualification. Performance qualification results of 20 consecutive days demonstrate the predetermined specification. Analytical instrument qualification and method validation relationship is also described by assay performed on haemodialysis concentrate. Good precision (<2%) and accuracy (95% - 105%), coefficient of determination (R<sup>2</sup>) greater than 0.9997 for potassium, magnesium and calcium linear curves, lower detection limits for all three ingredients and robust assay data as a part of validation studies conform the qualified AAS for its intended application.
Analytical instrument qualification (AIQ) is getting reliable and valid data from it for its intended application. AIQ is important in generating the quality data for analytical method validation and provides the contribution towards the quality analysis. AIQ involves the four discrete steps of qualification such as design qualification (DQ), installation qualification (IQ), operational qualification (OQ) and performance qualification (PQ). Atomic absorption spectrometer (AAS) is among the group C instruments where conformance is complex; highly method specific and the conformity bounds are determined by their application and full qualification process applies [
Standard solution of copper, cadmium, potassium, calcium, magnesium (1000 μg/mL) and nitric acid (AR grade) were purchased from Thermo Scientific, UK. Hallow cathode lamps of copper, manganese, mercury, potassium, calcium, magnesium and cadmium were parts of AAS as purchased from PG-Instruments, UK. Grade 1 quality water (0.01 μS/cm) was prepared in our own laboratory [
AAS unit with graphite furnace, compressor, chiller and eight turrets hallow cathode lamps (HCL) with deuterium background correction with bandwidth 0.2 - 1.0 nm and computerized automatic control over the main unit were the user requirements. These mentioned requirements which are easy to assemble, dismantle and cleaning approach to various components like hallow cathode lamps and graphite furnace.
Tracebility and linkage
Verification of software and hard ware products like compatibility of AAWin® (data station) for data processing and HCL, pressure control regulators for acetylene gas, argon gas and air respectively as defined in the functional requirements were documented. Electrical, mechanical and environmental specifications were also assured.
The operational functions of AAS such as wavelength accuracy and reproducibility, resolution, baseline stability, characteristics concentration or sensitivity, detection limit, characteristics mass and precision were determined and demonstrated the functions as expected. The parameters for all these operational functions are presented in
Wavelength correction was carried out by using the mercury (Hg) lamp with 0.2 nm bandwidth and records the peaks at 253.7 nm, 546.1 nm, 871.6 nm. The average value was calculated by using the relationship
The parameters for evaluation of performance qualification was same as that of operational qualification by running the system for 20 consecutive working days, all relevant information and data was recorded.
. Parameters for operational functions
Parameters | Wavelength accuracy and reproducibility | Resolution | Baseline stability | Characteristics concentration or sensitivity | Detection limit | Characteristics mass (GF) | Precision |
---|---|---|---|---|---|---|---|
HCL | Mercury | Manganese | Copper | Copper | Copper | Cadmium | Copper |
Lamp current (mA) | 3.0 | 2.0 | 3.0 | 5.0 | 5.0 | 5.0 | 5.0 |
Bandwidth (nm) | 0.2 | 0.2 | 0.2 | 0.4 | 0.4 | 0.4 | 0.4 |
Negative voltage (volt) | 300 | 300 | 300 | 300 | 300 | 300 | 300 |
Gas flow (acetylene) (mL/min) | 1200 | 1700 | 2000 | 2000 | 2000 | - | 2000 |
Flame height (mm) | 6.0 | 6.0 | 6.0 | 6.0 | 6.0 | - | 6.0 |
Flame position (mm) | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | - | 0.0 |
GF: Graphite Furnace.
AIQ and method validation are interlinked with each other, tested and defined specification as part of qualification confirmed that system is suitable for validation of a method. For the purpose of method validation system suitability tests is performed on quality control samples and compare its results with known values [
These qualification steps were performed at the site of Renacon Pharma (Pvt.) Limited, Lahore, Pakistan.
Technical specification like wavelength range (190 - 900 nm), deuterium background correction, bandwidth 0.2 - 1.0 nm with automatic scanning mode, data processing of measurement and read out mode of package unit and components were assured from manual provided by manufacturer. AAS in analytical laboratory with all accessories such as oil free compressor, reducing valves for argon, acetylene gases and chiller for graphite furnace were checked which are well fitted as part of user requirement specification and site acceptance test. These parameters gave assurance to DQ. As a part of IQ, environmental conditions were assured, AAS was installed in laboratory where temperature (<25˚C) and humidity (<50%) are well maintained, away from electromagnetic field, strong radiation, strong light, solvent vapors and chemical treatment room then configured the computer and connected peripheral devices. Verified the software data processing such as measurement in absorbance, concentration, transmittance, emission intensity and read out in continuous, peak height, peak area mode which is functional requirements. Data processing functions like multi-standard calibration method, standard addition method, interpolation method, integration time: 0.1 - 20 sec, sampling delay: 0 - 20 sec, no of standard samples 1 - 8, no of samples 0 - 100, slope, mean value, standard deviation (SD), relative standard deviation (RSD), correction coefficient, concentration value were also verified. Analytical results, instrumental and measurement parameters, signal profile and calibration curves all were stored in the computer hard disk confirmed the data storage functions. Wavelength accuracy was evaluated on three different wavelengths of mercury, the scanned peak in triplicate and standard wavelength are presented in
Wavelength reproducibility was performed at the interval of 6 hrs maximum and minimum value were reported, the difference between upper and lower value within the acceptance criteria. Resolution was reported by searching the peak at 279.5 nm using manganese HCL, three peaks were obtained at 279.46, 279.78 and 280.10 nm. The ratio of the valley energy values at first two peaks is 0.191 (n = 3) which is less than 0.30 as recommended by manufacturer. The resolution data is summarized in
Baseline stability was performed by running the blank solution (0.5%) using copper HCL at 324.7 nm and recorded the maximum and minimum absorption values at interval of 10 sec, reported the 30 consecutive readings. The difference between the average upper and lower values is 0.0017 which is in accordance with operation requirement less than 0.005 as manufacturer recommendation. The baseline stability is presented in
The characteristics concentration was found to be 0.062 µg/mL which is obtained by using the copper standard solution (0.5 µg/mL) and blank solution absorption measurement alternatively. The detection limit of AAS was measured by running the blank solution and absorption was measured in triplicate with 3 sec integration time and SD of 20 consecutive readings was calculated which is found to be 0.00118 and detection limit is 0.00354 µg/mL. The characteristics mass which is specific for graphite furnace mode of AAS measurement was found to be 1.41 pg after injecting 20 µL cadmium standard solution (0.1 µg/mL) while the user limit is 5.0 pg. The precision of AAS in both flame and graphite furnace mode was obtained by using copper and cadmium standard solution respectively and reported in % RSD. The % RSD is 0.532 for flame mode and 0.487 graphite mode. The results of characteristics concentration, detection limit, characteristics mass and precision data is reported in
. Wavelength accuracy and reproducibility
Wavelength accuracy | |||
---|---|---|---|
Standard wavelength (nm) | Wavelength obtained (n = 3) | Difference | |
253.700 | 253.683 | −0.017 | |
546.100 | 546.003 | −0.097 | |
871.600 | 871.630 | +0.030 | |
Wavelength reproducibility | |||
Time interval (6 hr) | |||
Maximum | Minimum | Difference | |
253.700 | 253.690 | 253.680 | 0.01 |
546.100 | 546.010 | 546.00 | 0.01 |
871.600 | 871.630 | 871.630 | 0.00 |
. Resolution studies
Standard value (nm) | Peaks obtained (nm) | Valley energy values ratio |
---|---|---|
279.5 | 279.46, 279.78, 280.10 | 14.9/78.2 = 0.190 |
279.5 | 279.46, 279.79, 280.12 | 15.0/78.0 = 0.192 |
279.5 | 279.47, 279.75, 280.08 | 15.0/78.0 = 0.192 |
Average | 0.191 |
. Characteristics concentration, detection limit, characteristics mass and precision data
Characteristics concentration (µg/mL) | Detection limit (µg/mL) | Characteristics mass pg | Precision % RSD |
---|---|---|---|
0.062 | 0.00354 | 1.41 | 0.532 (flame), 0.487 (graphite furnace) |
Baseline stability
All these parameters were performed as the part of OQ. PQ parameters were same as that of OQ but the AAS was run for consecutive 20 days, PQ results demonstrated that performance consistently meets pre-determined specifications. The PQ results average of 20 days are summarized in
For purpose of system suitability each standard of potassium, magnesium and calcium was run in triplicate and results are summarized in
766.5 nm, 422.7 nm and 285.2 nm absorption lines were selected for specificity studies; at these selected lines no spectral interference was observed. Repeatability and intermediate precision was performed on same and three consecutive days respectively. Six replications of each of standard (potassium: 2.00 µg/mL, magnesium: 0.75 µg/mL, calcium: 1.25 µg/mL) that is 100 percent of target concentration was performed low value of % RSD conforms the method precision. Precision results are reported in
A six point calibration curve was drawn which covers the 80% - 120% of target concentration in sample and linear regression equation was applied for limit of detection and limit of quantitation using the formula (LOD = yB + 3sB; LOQ = yB + 10sB) respectively. Coefficient of determination (R2) for three standards curves is greater than 0.9997. Parameters of linear regression equation, LOD and LOQ are presented in
Accuracy is reported in terms of % recovery studies, sample of haemodialysis concentrate was spiked with known concentration of standard solution of each content at low (0.10 µg/mL), medium (0.15 µg/mL) and high (0.20 µg/mL) levels and all the measurements are performed in triplicates. The results of recovery studies are in between 95% - 105% which in compliance with ICH guidelines. The recovery data is reported in
Purposeful changes in measuring parameters like integration time (0.3 sec - 0.2 sec), bandwidth (0.4 nm - 0.2 nm) and flame height (6.0 mm - 5.0 mm) was made for robustness and assay was performed. Robust data of performed assay (% mean ± SD) is reported in
. PQ results
Wavelength accuracy and reproducibility | <0.025 nm |
---|---|
Resolution | 0.197 |
Baseline stability | 0.0017 |
Characteristics concentration or sensitivity | 0.061 |
Detection limit | 0.00352 |
Characteristics mass (GF) | 1.42 |
Precision | 0.534 (flame), 0.489 (graphite furnace) |
. System suitability tests
Concentration | Potassium (n = 3) | Magnesium (n = 3) | Calcium (n = 3) | ||||||
---|---|---|---|---|---|---|---|---|---|
µg/mL | Std. 1 | Std. 2 | Std. 3 | Std. 1 | Std. 2 | Std. 3 | Std. 1 | Std. 2 | Std. 3 |
Theoretical | 1.60 | 2.00 | 2.40 | 0.50 | 0.75 | 1.00 | 0.60 | 1.25 | 2.00 |
Measured | 1.61 | 1.99 | 2.41 | 0.51 | 0.76 | 0.99 | 0.59 | 1.24 | 2.01 |
% RSD | 0.89 | 1.02 | 0.76 | 0.63 | 0.78 | 0.91 | 1.01 | 1.02 | 0.21 |
. Precision data
Concentration | Repeatability (n = 6) | Intermediate precision (n = 6) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
µg/mL | Potassium | Magnesium | Calcium | Day 1 | Day 2 | |||||
Potassium | Magnesium | Calcium | Potassium | Magnesium | Calcium | |||||
Theoretical | 2.00 | 0.75 | 1.25 | 2.00 | 0.75 | 1.25 | 2.00 | 0.75 | 1.25 | |
Measured | 1.99 | 0.76 | 1.26 | 2.01 | 0.74 | 1.26 | 2.02 | 0.74 | 1.26 | |
% RSD | 0.63 | 0.91 | 1.01 | 0.21 | 0.54 | 0.78 | 0.23 | 0.87 | 0.91 | |
. Linear regression equations, LOD and LOQ
Contents | Concentration (µg/mL) | Equation | R2 | LOD (µg/mL) | LOQ (µg/mL) |
---|---|---|---|---|---|
Potassium | 0.0 - 5.0 | y = 0.0852x + 0.0068 | 0.9998 | 0.0010 | 0.0034 |
Magnesium | 0.0 - 1.0 | y = 0.1478x + 0.0019 | 0.9998 | 0.0018 | 0.0060 |
Calcium | 0.0 - 2.5 | y = 0.0232x + 0.0063 | 0.9998 | 0.0042 | 0.0141 |
. Recovery studies
Spike level | Potassium % | Magnesium % | Calcium % |
---|---|---|---|
Mean ± SD | Mean ± SD | Mean ± SD | |
Low | 99.09 ± 0.32 | 99.69 ± 0.12 | 99.19 ± 0.02 |
Medium | 98.12 ± 0.11 | 100.19 ± 0.41 | 99.72 ± 0.91 |
High | 101.01 ± 0.34 | 99.01 ± 0.44 | 100.01 ± 0.94 |
. Robustness data
% Assay mean ± SD | ||
---|---|---|
Potassium | Magnesium | Calcium |
100.01 ± 0.09 | 99.83 ± 0.12 | 99.78 ± 0.56 |
PG-990 AAS of PG-Instruments, UK was qualified for its intended applications in pharmaceutical industry. Attaining the correct results, rapid identification and rectification of measurement related problems are well handled by qualified instrument. Qualification also provided the ground to develop and validate new analytical methods.