HPLC Method Adjustment USP way

Introduction and outcome

HPLC method adjustment is required during pharmaceuticals analysis. According to standard test procedure analysis can not be initiated if the HPLC system fails in the system suitability test. Now the question is why does the HPLC system fail in the system suitability test and what is the solution? This is a very critical situation for any analytical or QC professionals. That is why I decided to share my skill-based knowledge on this topic. In this article, I will discuss reasons for failing the system suitability test, the need of adjustment in the HPLC method, necessary and sufficient conditions under which adjustments can be made in the HPLC method, adjustments in the isocratic condition, adjustment in the gradient conditions, case studies and frequently asked questions. After reading this post all your doubts will be cleared and you will be able to apply more effectively during method development and routine analysis.

Table of Content

• Introduction and outcome
• Why does the HPLC system fail in the system suitability test?
• Need for adjustment in the HPLC method
• Necessary and sufficient conditions under which adjustments can be made in the HPLC method
• Adjustment in LC condition in Isocratic elution
  • Stationary phase (SP) adjustment
  • Stationary phase porosity adjustment
  • Column dimension (Length and particle size)
  • Flow rate adjustment
  • Mobile phase composition adjustment
  • pH adjustment
  • Buffer concentration adjustment
  • Flow rate of the mobile phase
  • Detector wavelength
  • Injection volume
• Adjustment in LC condition in Gradient elution
  • Adjustment in the Gradient time
  • Adjustment in column temperature
  • Adjustment in the mobile phase (composition and gradient)
• Conclusion
• FAQs

Why does the HPLC system fail in the system suitability test?

The following factors may lead to failure in the system suitability test and variation in the retention time (RTs), relative retention time (RRTs), co-elution of peak, and elution pattern:

• Laboratory temperature: Due to variation in the laboratory temperature there may be changes in mobile phase composition due to evaporation of the solvents
• Solvent quality: The purity of the solvent may affect the system suitability test
• Column life: Column life is inversely proportional to the number of injections. The new column performs and pass the system suitability test easily whereas an old column needs adjustment in the HPLC condition
• Column brand: Retention time (RT) and relative retention time (RRT) are not the same in all the columns of different makes containing the same stationary phase. In that case also adjustment in the HPLC method is required to fulfil the system suitability criteria
• Mobile phase: The life of the column depends upon the solvent, buffer concentration and the pH of the mobile phase. In some of the mobile phases, life of the column is longer whereas in some of the mobile phases life of the column is shorter. That is why adjustment in HPLC method is required to meet the system suitability criteria.
• Column brand: The same column of different brands gives different retention time for an analyte
• Column dimension and stationary phase particle size: SST may fail or RTs and RRTs may change while changing dimension or stationary phase particle size

Hence, HPLC condition must be adjusted to meet RTs, RRTs , SST criteria and elution pattern.

Need for adjustment in the HPLC method

Adjustment to the HPLC method is made when it fails in RTs, RRTs, SST criteria and elution patterns. When the specified column is not available as outlined in the method and an equivalent column is used in the analysis, in that case also method can fail in RTs, RRTs , SST criteria and elution patterns. This is why the HPLC condition is adjusted to meet the RTs, RRTs, SST criteria and elution patterns. Since, HPLC analysis is performed in isocratic and gradient conditions and therefore adjustments are applicable in both conditions.

Necessary and sufficient conditions under which adjustments can be made in the HPLC method

• SST criteria must be within the acceptance limit
• There must be the same elution pattern as in the monograph method
• All impurities must be detected and pass the acceptance criteria
• All impurities must fulfil sensitivity or quantitation limit acceptance criteria and
• Must pass in linearity and resolution test

HPLC method adjustment in Isocratic elution

Stationary phase (SP) adjustment

Changes or adjustments in the stationary phase are not allowed e.g. C18 stationary phase can not be used in place of the C8 stationary phase and vice-versa.

Stationary phase porosity adjustment

A change from total porous particle (TPP) to superficially porous particle (SPP) is permitted provided that the plate number (N) is -25% to 50% relative to the prescribed column

Column dimension (Length and particle size)

The adjustments in length of the column and particle size are permitted, provided that the ratio of the column length (L) and particle size (dp) remains constant or in the range between -25% to +50% of prescribed L/dp ratio.

Example: if you have a method in which in which column is C18,(300 x 4.6) mm, 10µm. Its L/dp will be 30 or L/dp =30. Hence you may use the following C18 columns:

• (150 x 4.6) mm, 5µm; L/dp = 30
• (100 x 4.6) mm, 3µm; L/dp = 33 or
• Any other column which can meet the L/dp criteria (range between -25% to +50% of prescribed L/dp) can also be used

Column Internal Diameter and Flow rate adjustment

The flow rate of the mobile phase should be adjusted when column particle size changes or the internal diameter of the column changes. The following equation should be used to adjust the flow rate:

F_{2 =} F₁ x [(dc₂² x dp₁/dc₁² x dp₂ )] (Equation-1)

Where:

• F₁: Flow rate indicated in the monograph (mL/minute)
• F₂: Adjusted flow rate (mL/minute)
• dc₁: Internal diameter of the column intended in the monograph
• dc₂: Internal diameter of the column used (mm)
• dp₁: Particle size indicated in the monograph(µm)
• dp₂: Particle size of the column used(µm)

Mobile phase composition adjustment

• The amount of minor component of the mobile phase can be adjusted ± 30% relative.
• But keep in the mind, the change in any component can not exceed ±10% absolute.

Binary mixture

Example-1: The specified ratio of the mobile phase is a mixture of water and methanol: 50 : 50. Hence, adjustment amount will be calculated in the following way:

30% of 50 is 50 x 30/100 = 15% absolute. But 15% exceeds the maximum permitted change of ±10% absolute. Therefore, the mobile phase ratio may be adjusted only within the range of 40 : 60 to 60 : 40.

Example-2: The specified mobile phase ratio is a mixture of water: methanol: 2 : 98. Hence, adjustment amount will be calculated in the following way:

30% of 2 is 2 x30100 =0.6% absolute. This is within the maximum permitted change of ±10% absolute in either component. Therefore, the mobile phase ratio may be adjusted only within the range of 1.4: 98.6 to 2.6 : 97.4.

Tertiary mixtures

The specified ratio of the mobile phase is a mixture of Water, Acetonitrile and methanol: 70 : 25 : 5. Hence, adjustment amount will be calculated in the following way:

Adjustment in acetonitrile: 30% of 25 is 25 x 30/100 = 7.5% absolute.This is within the maximum permitted change of ±10% absolute in either component. Therefore,this component may be adjusted within the range of 32.5% to 17.5% absolute

Adjustment in methanol: 30% of 5 is 5 x 30/100 = 1.5% absolute.This is within the maximum permitted change of ±10% absolute. Therefore, this component may be adjusted within the range of 6.5% to 3.5% absolute.

Please note that in all the cases sufficient amount of the first component is used to give a total of 100% or in other words some of all components (water, acetonitrile and methanol) must be 100.

Therefore maximum range will be 62.5 : 32.5 : 5 to 77.5 : 17.5 : 5 or 68.5 : 25 : 6.5 to 71.5 : 25 : 3.5 would meet the requirement

pH adjustment

The allowable adjustment of the pH of the aqueous component is ± 0.2pH units unless otherwise prescribed.

Example: If the specified pH is 7. Then it may be adjusted between 6.8 to 7.2

Buffer concentration adjustment

The allowable adjustment for the buffer concentration is ±10%

Example: If in a method concentration of KH₂PO₄ is 20mM. Then it will be adjusted between 0.18mM to 0.22mM

Flow rate of the mobile phase adjustment

An adjustment of the flow rate by ±50% is permitted.

Example: If the flow rate in any method is 1mL/minute. Then it will be adjusted between 0.5mL/minute to 1.5mL/minute

Detector wavelength adjustment

No adjustment is permitted

Injection volume adjustment

When the columns dimensions are changed , the following equation should be used for adjusting the injection volume:

V_inj2 = V_inj1 (L₂d_c2 ²)/(L₁d_c1 ²)

Where:

• V_inj1: Injection volume indicated in the monograph (µl)
• V_inj2: Adjusted injection volume i(µl)
• L₁: Column length indicated in the monograph (mm)
• L₂: Internal diameter of the column used (mm)
• d_c1: Particle size indicated in the monograph(µm)
• d_c2: Particle size of the column used (µm)

This equation will not be applicable to change in stationary phase from TTP columns to SPPs columns

The modified injection volume must pass the following parameters:

• SST criteria must be within the acceptance limit
• All impurities must be detected and full-fill sensitivity/QL acceptance limit
• Must pass in linearity and resolution test

HPLC method adjustment in Gradient elution

Since most of the analysis is performed in gradient mode and therefore adjustment in chromatographic condition in gradient mode needs more precautions than isocratic mode.

Adjustment in column parameters and flow rate

The adjustment in the following chromatographic parameters will be made same as in adjustment in LC condition in Isocratic elution

• Stationary phase
• Stationary phase porosity
• Column dimension (Length and particle size)
• Column internal diameter
• The flow rate of the mobile phase
• Adjustment in column temperature
• pH adjustment
• Concentration of salt adjustment
• Detector wavelength

Adjustment in the Gradient time

Column volume will be changed due to a change in column dimension.Since the gradient elution pattern depends upon the column volume and therefore gradient time must be modified. The following equation should be used to calculate the gradient time:

t_G2 = t_G1 x (F1/F2) [(L₂ x dc₂² )/L₁ x dc₁² )]

Where:

• t_G1: Gradient time given in the method
• t_G2: New gradient time
• F1: Flow rate given in the method
• F2: New flow rate and it will be calculated using equation
• L₁: Column length given in the method
• L₂: Length of the new column
• d_c1: Particle size indicated in the monograph(µm)
• d_c2: Particle size of the column used (µm)

The adjustment in conditions for gradient elution requires three steps:

• Adjust the column length and particle size according to L/dP
• Adjust the flow rate for changes in particle size and column diameter
• Adjust the gradient of each segment for changes in column length, diameter and flow rate

Adjustment in column temperature

The allowable adjustment in column temperature is ±5^oC

Example: If column temperature is 30^oC is mentioned in the method. Then adjustment can be done between 25^oC to 35^oC during analysis.

Adjustment in the mobile phase (composition and gradient)

The Adjustment in the mobile phase composition and gradient is allowed in the following conditions:

• The SST acceptance criteria must be full-filled
• The principal peak should elute within ±15% of the retention time obtained with the original condition
• The composition of the mobile phase and gradient is such that first peak should retain sufficiently and last peak must elute

Conclusion

This USP guideline on changes in chromatographic method is very helpful. This guiling is more suitable for isocratic methods compared to the gradient methods. This will be applicable only for the USP monographs . For In-house or method of other sources revalidation or mini validation must be performed and mini-validation must include all allowable changes. This is all about this article. Now I hope all your doubts have cleared and you can apply it more effectively during HPLC method development.

FAQs

Why some time HPLC fails in system suitability test?

System suitability test may fail due to improper washing of column, column degradation, error in mobile phase preparation, lab temperature and improper system equilibration

What are the allowable change in HPLC condition?

Column length and particle size can be adjusted but L/dp (where L is the length of the column and dp is the particle size of the column) should be between -25 to 50% of the original column. Stationary phase chemistry can not be changed. Flow rate can be adjusted to ±50% of the original flow rate. Keep in mind any adjustment must pass the system suitability test.

How much change is allowed in the HPLC method without revalidation? What are the allowable adjustment for USP?

Column length and particle size can be adjusted but L/dp (where L is the length of the column and dp is the particle size of the column) should be between -25 to 50% of the original column. Stationary phase chemistry can not be changed. The flow rate can be adjusted to ±50% of the original flow rate. Column temperature can be adjusted to ±5^oC and pH can be adjusted to 0.2 pH unit. Keep in mind any adjustment must pass the system suitability test.

Can these changes be made to In-house method?

No. In inhouse method changes will be made as per validation report.

Can these adjustments be allowed in all HPLC methods?

It will be applied to all HPLC monograph methods. It can not be applied in-house or other methods. Keep in mind any adjustment must pass the system suitability test.

Can above adjustment be applicable to all USP monograph?

Yes. Keep in mind changes must pass the system suitability test

Abbreviations

• L: Length of the column
• dp: Particle size of the stationary phase
• QC: Quality control
• RT- Retention time
• RRT: Relative retention time
• USP: United States pharmacopeia

References

• USP General chapter <621>
• Instrumental method of analysis; Willard, Merritt, Dean and Settle