method development and validation, quality based design for cyclophosphamide using hplc

QUALITY ASSURANCE, CLINICAL TRIALS & REGULATORY AFFAIRS

PJ4018

Francesco De luca

Development and validation of a new HPLC analytical

method for the determination of diclofenac in tablets Bushra Tuwfeeq Alquadeib

King Saud University, Riyadh, Saudi Arabia

Aim and Objectives

Faster analysis of Diclofenac Sodium in pharmaceutical formulations

Development of a new HPLC method for the evaluation of Diclofenac Sodium in tablets

Validation of method

BACKGROUND

Diclofenac is a widely used NSAID (nonsteroidal anti-inflammatory drug)

Treatment of rheumatoid arthritis, musculoskeletal injuries, and post surgery

analgesia

Water insoluble but quickly absorbed by the GI tract if used as the salt form

HPLC has already been used for the determination of Diclofenac in dosage forms but they require time and are expensive

Method Used

Different products selected randomly : Voltaren, Clofen, Voltaic, Rapidus and Rofenac

Chromatografic separation obtained using C18 as stationary phase and a mix of Orthophosphoric acid and Acetonitrile (35:65) as mobile phase

Lidocaine used as internal standard

Running time is 2 minutes

Validation

Fulfill ICH requirements on specificity, linearity, LOD, LOQ, accuracy, precision and robustness.

➢ Specificity

Evaluated to avoid interference from excipients

Placebo and standard solution have been used

No peaks close to the one from Diclofenac Sodium

➢ Linearity

Studied by injecting seven solutions with different concentration of Diclofenac Sodium

A calibration curve was created with each point representing the mean of six determination

Validation

➢ LOD and LOQ

Calculated based on the calibration curve

Value used have been standard deviation of response and slope of the calibration curve

LOQ was 3.9 ng/mL and LOD 13.1 ng/ml

➢ Precision

Six replicates of different concentrations were analysed on the same day to obtain intraday precision

The same procedure has been applied on three different days to obtain inter-day precision

The total precision has been measured using relative standard deviation (<2%)

➢ Robustness

Robustness was studied by changes in the method like using different pH of the mobile phase or wavelength

No big changes in the chromatogram showed this is a robust method for analysis.

Stability Studies

Stability tests have been carried on to test the stability of the sample in the auto sampler during the analysis

Temperature used have been 25°C, 4°C and -35°C for three weeks

Diclofenac Sodium has proven to be stable at 4°C and -35°C but a significant loss of active compound has been registered at 25°C

Results Obtained

New HPLC method for the analysis of Diclofenac Sodium

Faster than the previous ones which focused on acetonitrile as the main solvent

Could be used to estimate the concentration of Diclofenac Sodium in plasma or other biological fluids

Positives

Simple and quick to use analytical method

Validation to ensure that every aspect follows ICH guidelines

Short explanation of every parameter used for validation

Negatives

No direct comparison with data obtained from other analytical methods

Rationale behind the use of this mix of Acetonitrile/Methanol is not explained

No picture of the chromatograms obtained during specificity studies

Determination of apomorphine freebase in

sublingual tablets by proton nuclear magnetic

resonance spectroscopy

Li Tan, Shook F.Chin, Virginia W. Miner, Liang Dong, Suneel Gupta, Steven M.Fields

Aim and Objectives

Provide an analytical technique for the development of Apomorhine sublingual tablets

Development of a 1H NMR method to determine the quantity of Apomorphine freebase and Apomorphine chloride in tablets

Evaluation of specificity and linearity

BACKGROUND

Apomorphine hydrochloride is a dopamine agonist drug used for the treatment of Parkinson’s Disease

The marketed drug is used as a subcutaneous injection multiple times a day

Need for a non-invasive procedure to administrate Apomorphine

Development of sublingual tablets

Apomorphine freebase has high permeability with a plasma profile similar to the subcutaneous injection

The freebase form is unstable in aqueous solution but a mixture of salt and freebase in a solid formulation increases stability

The ratio between freebase form and chloride salt is critical for quality and efficacy

Previous quantification methods

➢ Titration Assay

Described by the US Pharmacopoeia in the Apomorphine monograph

Titration of HCl in the formulation

Cannot determine freebase content

➢ HPLC

Samples must be dissolved in an acidic solution

All Apomorphine gets protonated and it is not possible to distinguish between salt and freebase

➢ Titration + HPLC

Determine salt form by titration and then both forms together by HPLC

Long process that require use of multiple solvents

Development of a 1H NMR method

1H NMR techniques have already been used to determine equilibrium constants by analysis of the chemical shift of particular groups

In this study the chemical shift of NCH3 is considered as an indicator of the ionization state

The chemical shift of NCH3 is proportional to the percentage of freebase form and salt form

A calibration curve has been prepared with solutions containing different ratios of Apomorphine freebase and its chloride salt

The chemical shift of NCH3 in Apomorphine freebase is at 2.908ppm

Method discussion

The methyl amine group is protonated in Apomorphine salt form but it isn’t in its freebase

Chemical shift of the non protonated group is lower because of more shielded protons compared to the salt

Three different solvent systems have been studied:

• Deuterated water (D2O) : Apomorphine freebase is not soluble enough in this medium

• DMSO: proton shift is almost the same in the two forms

• Deuterated Methanol (CD3OD) : high solubility, high polarity, substantial change in the chemical shift

Samples analysed contained different percentages of the two forms :

1) 100% Apomorphine HCL

2) 100% Apomorphine Freebase

3) 75% Apomorphine Freebase (normal content of a sublingual tablet)

1H NMR Results

Method Validation

➢ Specificity

Excipients added to the mixture to evaluate possible interference

No significance change in the chemical shift

Robust method

➢ Linearity

Verify linearity to support future formulation development

Determined from 100% Apomorphine freebase to 0% freebase

Excellent linearity

Conclusion

A new 1H NMR method to analyse mixture of Apomorphine freebase and salt form

Faster and cheaper than previous ones

Specific and robust method that can be used for further development of more Apomorphine formulations

Positives

Improved analysis of a formulation still in development

Clear explanation of method and materials used

Good comparison of different NMR spectra

Negatives

Few information about the calibration curve

Specificity and linearity data have only been stated but no graph or diagram has been shown

Bibliography

Alquadeib, B.T. (2019) 'Development and validation of a new HPLC analytical method for the determination of diclofenac in tablets', Saudi Pharmaceutical Journal, 27(1), pp. 66-70. doi: //doi.org/10.1016/j.jsps.2018.07.020.

Tan, L., Chin, S.F., Miner, V.W., Dong, L., Gupta, S. and Fields, S.M. (2016) 'Determination of apomorphine freebase in sublingual tablets by proton nuclear magnetic resonance spectroscopy', Journal of pharmaceutical and biomedical analysis, 129, pp. 378-382. doi: S0731-7085(16)30362-4 [pii].