PHARMACEUTICAL: calibration of HPLC

escription

1.0    purpose:

1.1     to lay down the procedure for calibration of hplc.

2.0    scope.

2.1    this sop is applicable for the calibration of hplc’s in qc / ipqc laboratories.

3.0    responsibility

3.1    executive-qc

4.0    accountability

4.1     asst. manager-qc

5.0    procedure

5.1    follow the respective sop for operation and carry out the 
following tests for the calibration of a hplc system as applicable.

5.1.1 flow rate accuracy

5.1.2 gradient composition acuracy

5.1.3 injection volume precision

5.1.4 oven temperature/sample chiller temperature   

         accuracy

5.1.5 wavelength accuracy

5.1.6 precision /carry over

5.1.7 response linearity

5.2    flow rate accuracy:

5.2.1 flow rate accuracy shall be checked for 0.5 ml, 1.0ml and 2.0  ml/minute with

      the following chromatographic conditions.

                column: restriction capillary

                mobile phase: hplc grade water

       5.2.1.1 program the flow rate to 0.5 ml/minute and allow to stabilize.

        5.2.1.2 take a clean and dried measuring cylinder of 10ml capacity  

                and weigh accurately(w1).

         5.2.1.3 take a calibrated stop watch.

       5.2.1.4 introduce the capillary outlet into the measuring cylinder and 

                start the stop-watch simultaneously.

        5.2.1.5 stop the watch after collection of about 10 ml water in the  

                cylinder  and withdraw  the capillary outlet simultaneously.

    note the time elapsed as ‘t’.

       5.2.1.6 weigh the measuring cylinder containing water accurately

                (w2). calculate the weight of water collected (w).

        5.2.1.7 calculate the volume (v) of water using the specific gravity of

                water at  specified temperature  as given below. calculate the  

                flow rate  as follows;

                flow rate (ml/minute)= v/t

5.2.1.8    repeat steps 5.2.1.1 to 5.2.1.7 and determine the flow rate accuracy for 1.0 ml/min and 2.0 ml/min.

5.2.1.9    record the details as per format no. xxxxx

                                                    table 1
volume of 1 g of water at various temperatures

s.no

   temperature( 0c)

   volume (ml)

   1

   20

   1.0027

   2

   22

   1.0033

   3

   24

   1.0037

   4

   26

   1.0044

   5

   28

   1.0047

   6

   30

   1.0053

5.3    gradient composition accuracy

5.3.1    determine the gradient composition accuracy using the following chromatographic conditions.

5.3.1.1    for low pressure gradient system

pre-purge

wavelength: 265 nm

flow:         2.0 ml/min

                 stop-time: 10.00

                 solvent a: hplc grade water.

                 solvent b:0. 5% acetone in hplc grade water.

                 timetable:

                  time         solvent b

                   0.01        100.0

                   8.0    100.0

                   8.01        0.0

gradient run:                   

wavelength: 265 nm

solvent a: hplc grade water.

solvent b:0. 5% acetone in hplc grade water.

flow:         2.0 ml/min

stop-time: 25 + 10 minute’s pre-purge.

                                                 table2

                                              time table

   time

   solvent b (%)

   1.00

   0.0

   1.01

   100.0

   5.00

   100.0

   5.01

   90.0

   8.00

   90.0

   8.01

   50.0

   11.00

   50.0

   11.01

   6.0

   14.00

   6.0

   14.01

   5.0

   17.00

   5.0

   17.01

   50.0

   20.00

   50.0

   20.01

   0.0

   25.00

   0.0

5.3.1.1 prior to test gradient composition perform a pre-purge.

        5.3.1.2 perform the test in triplicate and measure the average height of every step

        5.3.1.3 re-scale the height and noise from absorbance to composition  (%b)  values.

        5.3.1.4 calculate the composition accuracy for every step as the absolute   

            difference between the rescaled measured step height  and the corresponding

            gradient composition values.

acceptance criteria    : ± 1.500%    

5.3.1.2    for high pressure gradient system

pre-purge

wavelength: 254 nm

flow:         1.0 ml/min

stop-time: 10.00

solvent a: hplc grade water.

solvent b:0. 3% acetone in hplc grade water.

                 timetable:

                  time         solvent b

                   0.01        10.0

                   10.0        10.0

gradient run:

wavelength: 254 nm

solvent a: hplc grade water.

solvent b:0. 3% acetone in hplc grade water.

flow:         1.0 ml/min

stop-time: 50 min.

                                                     table3

                                                 time table

   time

   solvent b (%)

   0.01

   10.0

   10.00

   10.0

   10.01

   50.0

   20.00

   50.0

   20.01

   90.0

   30.00

   90.0

   30.01

   100.0

   40.00

   100.0

   40.01

   0.0

   50.00

   0.0

5.3.1.1 prior to test gradient composition perform a pre-purge.

        5.3.1.2 perform the test in triplicate and measure the average height of every step

        5.3.1.3 re-scale the height and noise from absorbance to composition  (%b)  values.

        5.3.1.4 calculate the composition accuracy for every step as the absolute   

            difference between the rescaled measured step height  and the corresponding

            gradient composition values.

acceptance criteria    : ± 1.0%

5.4    injection volume accuracy

5.4.1    injection volume precision shall be determined for 10?l, 20 ?l, 50 ?l and 100?l injections.

                    chromatographic conditions:

                    mobile phase: hplc grade water.

                    column:  restriction capillary.

                    run time: 1 min

                    flow: 1 ml/min

    5.4.1.1 fill four vials with hplc grade water and cap them with  septum.

    5.4.1.2 identify the vials and accurately weigh each of the vials  separately.

    5.4.1.3 place the vials in the sample tray of the hplc system and program an

                                         injection  sequence as follows.

                                                    table3

   vial                           injection volume      repetitions

   vial1                                   10?l                           5

   vial2                                   20?l                           5

   vial3                                   50?l                           5

   vial4                                   100?l                          5

    5.4.1.4 after completion of the injection cycle weigh the vials  accurately and

          determine the average injection volume for each nominal volume using the

          volume of water per gram at specified temperature as included  in table 1.

                                      record the details as per format no. xxxxx

                                      acceptance criterion:  + 10.0% of programmed value.

note  :  injection volume accuracy is applicable only to hplc system

  having autosampler / autoinjector.

5.5    oven temperature/sample chiller temperature accuracy

5.5.1    oven temperature accuracy shall be checked at 300c, 400c and 500c.

5.5.2    sample chiller accuracy shall be checked at the temperatures 100c, 150c and 200c.

5.5.3    set the temperature of the oven /sample chiller to one of the above 

            mentioned values, insert the temperature probe  and allow to stabilize. 

            after stabilization monitor the temperature at intervals of 5 minutes and 

  note down three readings of the observed value. repeat the procedure 
for the  other  nominal values and record the details as per format no. 
xxxxx

    acceptance criterion:  + 20c of programmed value. 

5.6    wavelength accuracy

5.6.1 wavelength accuracy of uv detector

       chromatographic conditions:

       sample: 20 ?g/ml caffeine in water.

       run time: 7 min.

       wavelength program:

5.6.1.1 fill the flow cell with caffeine standard solution by injecting   

                directly  into  the detector inlet and start the program.

    5.6.1.2 record the peak response at various wavelengths as per

          format noxxxxx   

acceptance criterion: wavelength maximum at 273+ 1nm.

note : if the system has no wavelength programming option, than 
perform the test by selecting wavelength manually for each injection.

5.6.1    wavelength accuracy of pda detector

5.6.1.1    fill the flow cell with caffeine standard solution by 
injecting  directly into the detector inlet and start the wavelength 
scan from 190 to 400nm.record the details as per format no. xxxxx      

acceptance criteria: maxima at 205+ 1 nm and 273 + 1 nm.

5.7    injection precision / carry over

5.7.1    perform injection precision and carry over as follows.

    chromatographic conditions;

    column: restriction capillary

    flow: 1.00 ml/min

    run time: 1.00 min

    mobile phase: hplc grade water.

    injection volume: 20.0?l

    wavelength:273 nm

        5.7.1.1inject 20.0?l of mobile phase three times followed by six 20?linj. of 

        caffeine (20?g/ml) in water and again  inject one blank (mobile phase).

5.7.1.2 calculate the injection precision for area as the % rsd of caffeine

          standard peak responses.

5.7.1.3    calculate the injection carry over for area as peak 
response in the end blank injection with the peak response from the 
third blank injection subtracted as % of the response of the sixth 
caffeine injection. record the calibration details as per format no. 
xxxxx

        acceptance criteria:    precision     : rsd nmt 2.0%

                            carry over area: nmt 0.20%.

5.8    response linearity:

5.8.1    determine the detector response linearity by injecting 
caffeine standard solutions of concentrations 10?g/ml, 20?g/ml, 30?g/ml,
 50?g/ml and 100?g/ml in water.

chromatographic conditions:  same as 5.7.1         

5.8.1.1 inject caffeine standard solutions of the above said concentrations.

5.8.1.2 record the chromatogram and take the peak areas of responses as per

            format no. xxxxx

5.8.1.3    plot a linearity graph of concentration vs peak response areas and record      

             the details as per format no. xxxxx               

acceptance criterion: correlation coefficient nlt 0.999

5.9    frequency:

5.9.1    once in six months.

Due to modular nature of the

system, the operation of each

unit is checked properly.



Operation check on overall system

o

Confirm that the system

controller and work station

control each unit during

analysis and that the analysis

results meet the prescribed

criteria.



Software and Firmware check

Here Firmware checking is

conducted based on version display and

Software certificate of Compliance. The

Software and Firmware must be properly

managed and change procedures must be

properly clarified.

Any problems identified in O.Q must be

investigated and appropriate actions must be

taken. All such actions must be documented

and approved by higher authority.

Prior to implementing O.Q, check the

system configuration, determine the items to

be evaluated and record them in O.Q record

and have them approved.

Performance Qualification (PQ):

The objective is to ensure that the

instrument is performing within specified

limits. Hence documented verification that

the equipment and ancillary systems, as

connected together, can perform effectively

and reproducibly based on the approved

process method and specifications.

The PQ represents the final qualification of

equipment or system. This incorporates a

range of testing to simulate your production

process options and provide assurance that

systems and operating documentation are

capable of subsequent process validation

activities. It is used to establish and or

confirm;

Definition of performance criteria and

test procedures.

Selection of critical parameters, with

predefined specifications.

Determination of the test intervals, e.g.,

(a) - Everyday.

(b) - Every time the system is used.

runs.

Define corrective actions on what to do if

the system does not meet the established

criteria.

Definition of Calibration: ICH

The demonstration that a particular

instrument or device produces results within

specified limits by comparison with those

produced by a reference or traceable

standard over an appropriate range of

measurements.

Calibration of HPLC:

Various Calibration

parameters are:



Flow rate accuracy



Injector accuracy



System Precision



Wavelength accuracy



Detector linearity



Injector linearity



Gradient Performance Check



Column oven temperature accuracy

Flow Rate Accuracy:

Prime all the solvent lines with Milli Q

water.

Set the flow rate to 0.500 ml/m.

Wait for about 15 m to stabilize the

tem and ensure that the pressure is stable.

Insert the outlet tubing into a 10 ml

volumetric flask and start the stop watch

simultaneously.

Stop the stopwatch when the lower

meniscus reaches the 10 ml mark on the

flask.

Record the elapsed time.

Similarly check the flow for 1.0 ml/m

and 2.0 ml/m.

Acceptance criteria:

The time taken to

collect the water should be with in ± 2.0% of

the actual value.

Gowrisankar

et al

/ J Biomed

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Res.,

Vol

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2010,89-99

Relationships

Table 1:

I.Q And O.Q And P.Q Report

System Name:

System ID No.:

Installation site:

Performer_______________Signature _______________Date:______

Reviewer_______________Signature________________Date:______

Manager_______________Signature________________Date:______

Table 2:

Flow Rate Accuracy

Set Flow

Actual time required to collect

up to the mark in m

Acceptance criteria (in m)

0.5 ml/m

1.0 ml/m

2.0 ml/m

20.0

10.0

5.0

19.6 – 20.4

9.8 – 10.2

4.9 – 5.1

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et al

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2010,89-99

Table 3:

Chromatographic Conditions for

System Precision

Column

Flow rate

Detection

injection volume

Run time

Column oven temperature

Mobile phase

,150mm×4.6 mm,5μm

1.0 ml/m

UV at 272 nm

20 μl

15 m

25°C ± 2°C

Water: Methanol (70:30v/v)

Injector Accuracy:

Connect the pump and detector inlet

with union.

Prepare mobile phase consisting of a

mixture of water and Methanol (70:30 v/v)

Set a flow rate of 0.5 ml/m and a run

time of 1 m.

Set the column temperature at 25±

2°C.

Fill a standard HPLC vial to 2/3

with

Milli-Q water. Seal the vial properly with a

cap.

Weigh the vial and record the weight

as W

grams.

Place the vial in the chromatographic

system and perform 6 injections of 50μl

volume from this vial.

Weigh the vial again and note the

weigh after the injections as W

grams.

Calculate the mean volume injected per

injection as follows:

Mean injected volume (μl) = (W

– W

)

×100/6

Acceptance criteria:

The mean injected

volume should be 50.0±1.0 μl.

System Precision:

Standard Preparation

: Accurately weigh

and transfer about 60mg of Caffeine into a

100ml volumetric flask. Dissolve and dilute

to the volume with mobile phase. Transfer

10ml of this solution into a 100ml

volumetric flask and dilute to the volume

with mobile phase.

Procedure:

Inject blank, followed by

standard preparation in 6 replicates. Note

down the areas and retention times.

Now calculate the %RSD of retention time

and peak areas for 6 replicates injections.

Acceptance criteria:

The %RSD of

retention time & peak area should be <1.0%.

Wavelength Accuracy:

Procedure:

Create and instrument method

with a wavelength in nm and inject blank,

followed by Standard preparation and note

down the height or absorbance.

Acceptance criteria:

The maximum

absorbance should be ±2nm.

PDA Detector Accuracy:

Select 3D mode and set the wavelength

range as 200-400nm.Inject 20 μl of standard

preparation once into the chromatographic

system. Extract and record the

chromatograms at wavelengths of 202 to

208nm with an interval of 1nm and at 269 to

275 nm with an interval of 1nm.Note down

the height or absorbance.

Acceptance criteria:

The maximum

absorbance should be at 205±2nm and

272±2nm.

Gowrisankar

et al

/ J Biomed

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and

Res.,

Vol

2 (2),

2010,89-99

Detector Linearity:

Standard Preparation:

Accurately weigh

and transfer about 60mg of Caffeine into a

100ml volumetric flask. Dissolve and dilute

to the volume with mobile phase.



Detector linearity solution 1(0.06

mg/ml):

Transfer 10ml of Standard

Preparation into a 100ml volumetric flask

and dilute to the volume with mobile phase



Detector linearity solution 2(0.048

mg/ml):

Transfer 8ml of Standard

Preparation into a 100ml volumetric flask

and dilute to the volume with mobile phase.



Detector linearity solution 3(0.03

mg/ml):

Transfer 5ml of Standard

Preparation into a 100ml volumetric flask

and dilute to the volume with mobile phase.



Detector linearity solution 4(0.24

mg/ml):

Transfer 4ml of Standard

Preparation into a 100ml volumetric flask

and dilute to the volume with mobile phase.



Detector linearity solution 5(0.012

mg/ml):

Transfer 2ml of Standard

Preparation into a 100ml volumetric flask

and dilute to the volume with mobile phase.

Procedure:

Inject blank, followed by

Detector linearity solutions and record the

peak responses of Caffeine standard plot

between the concentration Vs the peak

responses.

Acceptance criteria:

The plot should be

linear and regression coefficient (R

) should

not be less than 0.99.

Injector Linearity:

Standard Preparation:

Accurately weigh

and transfer about 60mg of Caffeine into a

100ml volumetric flask. Dissolve and dilute

to the volume with mobile phase.

Transfer 10ml of Standard Preparation into a

100ml volumetric flask and dilute to the

volume with mobile phase.

Procedure:

Inject 5 μl of the mobile phase

as blank injection.

Inject 5 μl, 10 μl, 20 μl, 50 μl and 80 μl of

the Standard Preparation and record the peak

areas. Plot a curve for the volume injected

Vs peak area.

Acceptance criteria:

The plot should be

linear and regression coefficient (R

) should

not be less than 0.99.

Gradient performance check:

Add 5ml of acetone to 1000ml of methanol

filter and degas. Connect the pump and

detector inlet with union. Set the detector

wave length at 254 nm.

Place Channels A and C in methanol and

channel B and D in 0.5% acetone in

methanol. Set binary gradient with a total

flow rate of 2.0ml/m.Set gradient program

as shown below for channels A, B and C, D

individually.

Table 4:

Gradient Performance Check:

Purge all the channels at a flow rate of

2ml/m for about 5 m. Set the flow rate at

2.0ml/m and wait until the base line is

stable. Set the gradient profile for A and B

Time

(m)

A or C

(Methanol)

B or D (0.5%

Acetone in

Methanol)

Initial

4.00

4.01

8.00

8.01

12.00

12.01

16.00

16.01

20.00

100.0

90.0

100.0

75.0

100.0

0.0

10.0

0.0

25.0

0.0

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2010,89-99

and run the gradient profile by injecting

“0.0”volume of methanol.

Record the height of the peaks. Consider the

height of the peak resulting from B at 100%

concentration as 100 and calculate the

percentage height of other peaks. Perform

the gradient performance check similarly for

channels C and D.

Consider the height of the peak resulting

from D at 100% concentration as 100 and

calculate the percentage height of other

peaks.

Calculations:

Height (%) of B/D =Height of B/D peak

×100/Height of full scale peak

Acceptance criteria:

The calculated

percentage composition (Height (%) should

be with in±1.0% of the set composition.

Column Oven Temperature Accuracy:

It is evaluated with a calibrated digital

thermometer at 30°Cand 60°C .Place the

thermometer probe in the column oven and

set the column oven temperature at

30°C.Wait till the temperature stabilizes.

Record the temperature displayed on the

thermometer. Similarly performs the column

oven temperature accuracy test at 60°C.

Acceptance criteria:

The resulting oven

temperature from the thermometer display

should be within ±2°C of the set

temperature.

NOTE:

For oven Temperature Accuracy,

Chromatographic conditions and mobile

phase refer to system precision test.

Calibration of Gas Chromatogrphy:

Various Calibration parameters are:



Flow rate accuracy



Column oven temperature accuracy



System precision



System precision for head space auto

sampler



Detector linearity



Detector noise and drift test

Flow rate accuracy:

Connect the digital flow meter to the

detector outlet port.

Set the carrier gas (Helium) flow and

wait till it reaches the set flow.

Note the observed flow in replicate.

Repeat the procedure for other carrier

gases such as Hydrogen and Air.

Record the result in GC calibration

protocol.

Acceptance criteria:

The flow rate of

carrier gas should be

±10% of set flow.

Table 5:

Flow Rate Accuracy:

S.No. Carrier

gas

Acceptance

criteria in

ml/m

1. Helium

125

2. Hydrogen

3. Air

400

Column Oven Temperature Accuracy:

Connect the column to the detector port.

2. Place the thermometer probe in the

column oven and set the column oven

temperature at 40°C.Wait till the

temperature stabilizes.

Note the observed temperature as read

by the probe in triplicate over a period of 10

Repeat the procedure for 100°C, 150°C

and 190°C.

Acceptance criteria:

The resulting oven

temperature from the thermometer display

should be within ±2°C of the set temperature

System Precision:

Preparation of Standard solution:

Transfer 20 ml of Methanol, Ethanol and

Acetone into 100ml volumetric flask and

make up with Ethyl acetate

Procedure:

Inject blank, followed by

Standard preparation in 6 replicates. Note

down the areas and Retention times.

Gowrisankar

et al

/ J Biomed

Sci

and

Res.,

Vol

2 (2),

2010,89-99

Table 6:

Chromatographic Conditions for System Precision

Column 30m

× 0.32mm,1.8μ,DB-624

Detector

Flame ionization detector

Injector temperature

180°C

Detector temperature

250°C

Flow mode

Pressure

Carrier Gas flow rate Helium

25 kpa

Oven program

50°C(hold 5 m) raise to 10°0C

Split ratio

1:10

Injection volume

0.2 μl

Hydrogen flow

40 ml/m

Air flow

400 ml/m

Table 7:

Chromatographic Conditions For Head Space Auto Sampler

Column 30m × 0.32mm,1.8μ,DB-624

Detector Flame

ionization

detector

Injector temperature

220°C

Detector temperature

260°C

Flow mode

Pressure

Carrier Gas flow rate Helium 25 kpa

Oven program

40°C(hold 5 m) raise to 200°C(hold 5 m)

Split ratio

1:10

Injection volume

0.2 μl

Hydrogen flow

40 ml/m

Air flow

400 ml/m

Table 8:

Head Space Conditions

Vial equilibrium

22 m

Vial pressure

0.5 m

Loop fill

0.5 m

Loop equilibrium

0.05 m

Inject 1.00

GC cycle time

38 m

Oven temperature

80°C

Loop temperature

100°C

Vial pressure

10.8 psi

Acceptance criteria:

The %RSD of

retention time should be not more than

1.0%& peak area should be not more than

5.0%.

System precision for head space auto

sampler:

Preparation of standard solution:

Prepare

a standard mixture solution by taking

Methylene dichloride (0.6g), Chloroform

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2010,89-99

(0.06g),Trichloroethane (0.08g),1,4-Dioxane

(0.38g) in 50ml volumetric flask containing

about 40ml of Dimethyl formamide.Finally

makeup to volume with DMF(Solution-A).

Procedure:

Take 0.5 ml of standard

solution-A in 6 different vials and seal with

septum, then magnetic caps and crimp. Place

these vials on head space sampler; prepare a

blank vial also. Load the vials in head space

sampler tray. Blank vials followed by the

standard vials.

Acceptance criteria:

The %RSD of

retention time should be NMT 1.0%& peak

area should be NMT 15.0%.

Detector linearity:

Preparation of standard solutions:



Detector linearity solution A:

Transfer

10ml of each Methanol, Ethanol and