4.10 Periodic Inspections of Systems
4.10.7 Carryover Test
Checks the residual sample level based on the area ratio of peaks obtained when a blank sample is injected after injecting a concentrated sample.
4.10.2 Absorbance Linearity Test
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Scope of Inspection
Verifies the absorbance linearity of the instrument based on the height of absorbance peaks detected when an aqueous caffeine solution is injected.
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Parts Required for Inspection
Part Name Description
Sample Set of 5 aqueous caffeine solution concentrations Flow Restrictor 4 m × 0.1 mm I.D. and 2 m × 0.5 mm I.D.
Mobile Phase Water (HPLC grade)
1.5 mL Sample Vials Uses five 1.5 mL vials. Equivalent containers may also be used.
1.5 mL Sample Vial Plate For holding 1.5 mL sample vials.
LabSolutions Data Processing Unit
Contents in Set of 5 Aqueous Caffeine Solution Concentrations
Name Solution 1 Solution 2 Solution 3 Solution 4 Solution 5
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Inspection Procedure
1 Connect the flow restrictor between the injector outlet tube and the detector.
2 Specify parameter settings for each instrument.
Item Setting Value
Quick Parameter
Settings Analysis Type ISO
Solvent Delivery
Pump Flowrate 0.5 mL/min
Autosampler
Rinse mode No rinsing
Sample speed 5 µL/sec (with 50 µL sample loop installed) 15 µL/sec (with other sample loop installed) Cooler temperature 25 ℃
Injection Volume 50 µL (base injection volume, changeable as necessary)
Column oven Oven Temperature 40 ℃
UV Detector
Wavelength 273 nm
Sampling Rate 20 msec
Response 0.1 sec (set automatically) Cell temperature 40 ℃
Data Processing Unit
WIDTH 2
SLOPE 20000
DRIFT 0
T.DBL 1000
MIN.AREA 20000
STOP.TM 2.0 min (changeable as necessary)
3 Start pumping the water.
4 Determine the injection volume by measuring at solution 5 (60.0 mg/L). Adjust the injection volume so that the peak height is in the range of 1.000-1.500 AU.
If the absorbance for solution 5 exceeds 1.5 AU, reduce the injection volume setting.
If the absorbance for solution 5 does not reach 1.0 AU, remove the 2 m × 0.5 mm I.D. flow restrictor and only use the 4 m × 0.1 mm I.D. flow restrictor.
5 After the baseline stabilizes, successively inject the samples (solutions 1 to 5).
6 Calculate the correlation coefficient contribution rate (R
2) based on absorbance peak heights.
Control Criteria 0.9990 or higher
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Corrective Actions if Linearity Does Not Satisfy Control Criteria
Cause Corrective Actions
The flow cell contains bubbles.
Implement measures to eliminate the bubbles.
4.3 Flow Cell Inspection/Cleaning in the SPD-40 CL Instruction Manual
The flow cell is contaminated.
Rinse the cell.
4.3 Flow Cell Inspection/Cleaning in the SPD-40 CL Instruction Manual
Mirror or window plate degradation has occurred.
Recalibrate linearity and perform optical system maintenance. (Contact a Shimadzu representative.)
4.10.3 Noise/Drift Test
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Scope of Inspection
Checks that noise and drift values satisfy specified criteria.
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Parts Required for Inspection
Part Name Description
Data Processing Unit LabSolutions
Flow Restrictor*1 4 m × 0.1 mm I.D. and 2 m × 0.5 mm I.D.
Mobile Phase*2 Purified water (HPLC grade or equivalent)
*1 The flow restrictor is removed for system configurations that result in abnormal pump pressures at the specified flowrate.
*2 Be sure to use a degassing unit to degas mobile phases.
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Inspection Procedure
1 Connect the flow restrictor between the injector outlet tube and the detector.
2 With the flow channels filled with degassed water, pump the water at a flowrate of 1.000 mL/min.
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3 Specify detector settings.
• SPD-40 CL absorbance detector:
Set the detector wavelength to 250 nm and the [AUX RANGE] setting to 1.0 AU/V.
4 For photodiode array detectors, also specify the multi-chromatogram setting in LC workstation data processing parameter settings.
Display: Enabled; Wavelengths: 250 nm and 600 nm; Bandwidth: 4 nm (±4 nm); and Ref correction: OFF
5 Specify data acquisition sampling values.
• SPD-40 CL absorbance detector:
Set sampling to 1000 ms and the response to 2 seconds. (Response is set automatically.)
6 After the instrument stabilizes, record the baseline for 15 minutes. Print and attach the results.
(Measure the baseline fluctuation at the point the instrument stabilized and record is as the drift/noise value.)
Acquire data at the same time as noise and drift tests. Calculate measurement results in compliance with ASTM methods.
Cell SPD-40
(250 nm)
Control Criteria
Standard Conventional
Noise ≦ 0.04 mAU Drift ≦ 0.50 mAU/h UHPLC
Semi-micro
Noise ≦ 0.06 mAU Drift ≦ 1.00 mAU/h
If the drift measurement results exceed the control criteria, confirm whether or not room temperature fluctuations satisfy the max. 2 ℃ condition.
Noise Evaluation Method
Divide 15 minutes of data into 30-second intervals.
Measure the noise band for each interval.
Determine noise as the average of noise bands for all 30 intervals. (Refer to: ASTM E1657-98)
Drift Evaluation Method
The method of calculating the drift value differs between automatic and manual calculations.
When an automatic calculation is performed using a workstation, the drift value is obtained by calculating the slope of the approximate straight line by the least-squares method from the set interval data, and converting it into the amount of change per 1 hour to obtain the drift (AU/h or RIU/h).
• In the case of manual calculation
15 minutes of data from the start point to the end point are connected, and the baseline fluctuation is calculated from that slope. The amount of baseline fluctuation over 15 minutes is multiplied by 4, and this is taken as the amount of
change over 1 hour, which is taken as drift (AU/h or RIU/h).
Determine drift (AU/h) as the fluctuation within an hour of data, calculated as four times the baseline fluctuation for the 15-minute period.
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3 Specify parameter settings as indicated below.
Solvent Delivery System
Pumping Mode [B.GE] or [LP.GE]
T.Flow
1.000 mL / min (Mixer volume under 0.5 mL)
2.000 mL / min (Mixer volume more than 0.5 mL)
COMP 0.45 (water)
LPGE Cycle Mode
(only for LPGE) 0: Standard
Absorbance Detector
Wavelength 273 nm for Caffeine solution.
265 nm for Acetone solution.
Sampling Rate 500 ms
Response 0.5 sec (set automatically)
AUX RANGE 1.0 AU/V
4 After specifying the above settings, set B.Conc to 50 % to check baseline stability by pumping at a constant flowrate.
5 Next, set B.Conc to 0 % to check baseline stability by pumping at a constant flowrate, adjust the zero point, and execute the gradient program indicated in Supplemental Instructions.
6 Move the cursor over the measured chromatogram to measure the signal level at the next concentration.
0 % (B.Conc = 0), 10 % (B.Conc = 10), 50 % (B.Conc = 50), 90 % (B.Conc = 90), 100 % (B.Conc = 100)
Measure the signal level when the baseline stabilizes.
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7 Measure the actual concentrations as described below.
Actual concentration at 10
% (%) =
(B.Conc 10 level) - (B.Conc 0 level) (B.Conc 100 level) - (B.Conc 0 level) ×100
Actual concentration at 50
% (%) =
(B.Conc 50 level) - (B.Conc 0 level) (B.Conc 100 level) - (B.Conc 0 level) ×100
Actual concentration at 90
% (%) =
(B.Conc 90 level) - (B.Conc 0 level) (B.Conc 100 level) - (B.Conc 0 level) ×100
8 For low-pressure gradient systems, test gradient performance for C and D in the same manner.
Control Criteria
(For HPGE system)
±1.0 % of the setting value (For LPGE system)
±2.0 % of the setting value Supplemental Instructions
Prepare and execute the following gradient program.
High-pressure Gradient Program
TIME FUNC VALUE
0.00 B.Conc 0.0
0.01 B.Conc 100.0
10.00 B.Conc 100.0
10.01 B.Conc 90.0
15.00 B.Conc 90.0
15.01 B.Conc 50.0
20.00 B.Conc 50.0
20.01 B.Conc 10.0
25.00 B.Conc 10.0
25.01 B.Conc 0.0
30.50 Stop
Low-pressure Gradient Program for A and B
TIME FUNC VALUE
0.00 B.Conc 0.0
0.01 B.Conc 100.0
10.00 B.Conc 100.0
10.01 B.Conc 90.0
15.00 B.Conc 90.0
15.01 B.Conc 50.0
20.00 B.Conc 50.0
20.01 B.Conc 10.0
25.00 B.Conc 10.0
25.01 B.Conc 0.0
30.50 Stop
Low-pressure Gradient Program for C and D
TIME FUNC VALUE
0.00 D.Conc 0.0
0.00 C.Conc 100.0
0.01 D.Conc 100.0
0.01 C.Conc 0.0
10.00 D.Conc 100.0
10.00 C.Conc 0.0
10.01 D.Conc 90.0
10.01 C.Conc 10.0
15.00 D.Conc 90.0
15.00 C.Conc 10.0
15.01 D.Conc 50.0
15.01 C.Conc 50.0
20.00 D.Conc 50.0
20.00 C.Conc 50.0
20.01 D.Conc 10.0
20.01 C.Conc 90.0
25.00 D.Conc 10.0
25.00 C.Conc 90.0
25.01 D.Conc 0.0
25.01 C.Conc 100.0
30.50 Stop
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4.10.5 Reproducibility Test
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Scope of Inspection
This inspection involves actually performing an analysis to confirm that data with good reproducibility is obtained.
The result is determined based on the relative standard deviation (coefficient of variation) of the retention time and peak area values for each peak.
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Parts Required for Inspection
Part Name Remarks
Flow Restrictor*1 4 m × 0.1 mm I.D. and 2 m × 0.5 mm I.D.
Mobile Phase*2 Water (HPLC grade)
Sample
Aqueous caffeine solution (20 mg/L, included in P/N 228-67619-41 set of 5 caffeine concentrations) or a sample prepared as follows:
1 Weigh 20 mg anhydrous caffeine and place it in a 100 mL volumetric flask. Dissolve with water to make 100 mL.
2 Place 1 mL of the caffeine solution in a 10 mL volumetric flask and add water to make 10 mL.
1.5 mL Sample Vials Two sample vials are used. The sample vials indicated on the left or equivalent may be used.
1.5 mL Sample Vial Plate For holding 1.5 mL sample vials.
LabSolutions Data Processing Unit
*1 The flow restrictor is removed for system configurations that result in abnormal pump pressures at the specified flowrate.
*2 Be sure to use a degassing unit to degas each mobile phase.
1 Connect the flow restrictor between the injector outlet tube and the detector.
2 Rinse the flow channels with an appropriate solvent.
3 Specify parameter settings for inspection.
Parameter Setting Values for Inspections
Item Setting Value
Quick Parameter
Settings Analysis Type ISO
Solvent Delivery
Pump Flowrate 0.5 mL/min
Autosampler
Rinse mode No rinsing
Sample speed 5 µL/sec (with 50 µL sample loop installed) 15 µL/sec (with other sample loop installed) Cooler temperature 25 ℃
Injection Volume 10 µL Column oven Oven Temperature 40 ℃
UV Detector
Wavelength 273 nm
Sampling Rate 20 msec
Response 0.1 sec (set automatically) Cell temperature 40 ℃
Data Processing Unit
WIDTH 2
SLOPE 20000
DRIFT 0
T.DBL 1000
MIN.AREA 20000
STOP.TM 2.0 min (changeable as necessary)
4 Place 1.0 mL of water in one sample vial and seal it with a new septum.
"2.5.2 Placing Samples in the Autosampler"
5 Place 1.0 mL of sample in another sample vial and seal it with a new septum.
"2.5.2 Placing Samples in the Autosampler"
6 Start adjusting the column oven temperature and start delivering solvents with the solvent delivery unit.
Confirm that liquid is flowing from the detector outlet tube and no liquid is leaking from any connections.
7 Monitor the baseline. Once the baseline is stable, adjust the detector zero-point.
8 Inject 10 µL of water and confirm that no peaks are observed.
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9 Inject and analyze 10 µL of the sample successively six times.
10 Calculate the relative standard deviation (coefficient of variation) for the retention time and peak area values based on the six analysis results.
: Number of analyses
: Retention time (or peak area) of individual peaks
: Mean value of X1...Xn : Standard deviation : Coefficient of variation
Control Criteria
Peak area CV % is within 1.0
%Retention time CV % is within 0.5 %
4.10.6 Injection Linearity Test
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Scope of Inspection
This inspection involves actually performing an analysis to confirm that data with good injection volume linearity is obtained.
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Parts Required for Inspection
Part Name Remarks
Flow Restrictor*1 4 m × 0.1 mm I.D.
Mobile Phase*2 Purified water (HPLC grade or equivalent)
Sample Aqueous caffeine solution (20 mg/L)
1.5 mL Sample Vials Uses one sample vial. The sample vials indicated on the left or equivalent may be used.
1.5 mL Sample Vial Plate For holding 1.5 mL sample vials.
LabSolutions Data Processing Unit
*1 The flow restrictor is removed for system configurations that result in abnormal pump pressures at the specified flowrate.
*2 Be sure to use a degassing unit to degas each mobile phase.
1 Connect the flow restrictor between the injector outlet tube and the detector.
2 Specify the same parameter settings as for the reproducibility test.
3 For five injection volumes, perform one analysis per volume.
Use the following 5 injection volumes.
Standard cell 2.0 µL, 5.0 µL, 10.0 µL, 20.0 µL, and 50.0 µL
4 Calculate the correlation coefficient contribution rate (R
2) based on peak area results.
Control Criteria 0.9990 or higher
4.10.7 Carryover Test
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Scope of Inspection
This validation inspects the carryover based on the measured peak area ratio.
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Parts Required for Inspection
Part Name Remarks
Flow Restrictor*1 4 m × 0.1 mm I.D. and 2 m × 0.5 mm I.D.
Mobile Phase*2 Water (HPLC grade)
Sample Aqueous caffeine solution (50 mg/L and 250 mg/L) 1.5 mL Sample Vials Uses three sample vials. The sample vials indicated on
the left or equivalent may be used.
1.5 mL Sample Vial Plate For holding 1.5 mL sample vials.
LabSolutions Data Processing Unit
*1 The flow restrictor is removed for system configurations that result in abnormal pump pressures at the specified flowrate.
*2 Be sure to use a degassing unit to degas each mobile phase.
1 Connect the flow restrictor between the injector outlet tube and the detector.
2 Specify the same parameter settings as for the reproducibility test and injection volume linearity test.
Set up the analysis method as shown below.
Parameter Setting Values for Inspections
Item Setting Value
Data Processing Unit
Algorithm i-PeakFinder
Detection Threshold 10
Min. Height 10
Minimum Half Width 10
3 Add 1 mL of 50 mg/L sample and 1 mL of 250 mg/L sample in each of two sample vial and seal them with a septum.
"2.5 Placing Samples"
4 Add 1 mL of water in one other sample vial and seal it with a septum.
"2.5 Placing Samples"
5 Place the sample vials in the sample plate and place the plate on the sample rack.
6 Inject 10 µL of blank water.
8 Inject 10 µL of 250 mg/L aqueous caffeine solution.
9 Inject 10 µL of blank water.
10 Record the peak area value obtained for blank water in step 9 and five times the peak area value obtained for the 50 mg/L aqueous caffeine solution in step 7.
11 Calculate the proportion (%) of the blank sample peak area value relative to the corresponding peak area for the 250 mg/L aqueous caffeine solution injected.
Carryover = (Blank peak area / 50 mg/L aqueous caffeine solution peak area × 5) × 100 Carryover ≦ 0.100 %