Method Development in Reversed Phase HPLC

Method development in HPLC is a challenging and interesting field of study. A number of factors need to be addressed in any method development exercise. We will deal with them on an individual basis in this chapter. 

The majority of analyses performed by HPLC in forensic science can be covered by using RP (reversed phase)-HPLC in one form or another. Column chemistry has advanced significantly, thus expanding the scope of such a technique. As a result of this, we have limited most of our method development discussions to reflect this. Additional texts covering method development for alternative separation modes can be found in the Further Reading section at the end of this chapter. 

When a method for HPLC is developed, it should be noted that each of the parameters that will be discussed is in no way isolated from one another and that any changes in the process should be made in a systematic manner. Automated systems are available to assist with the method development process and examples of these will be discussed as well. However, before any software programme is used, it is necessary to have an understanding of the processes involved in order to ensure that a correct and valid outcome is achieved. 

The three most important things that need to be addressed are 

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Fig. 2. Sequence of partly automated method development in reversed-phase HPLC.

The global optimum for a separation problem can be better found and wdth fewer manual tasks than previously. Method development should less frequently end in an inextricable cul-de-sac. With HEUREKA, the earlier ideas of automating method development in reversed-phase HPLC have been consistently developed further and put into practice [5]. 

Snyder L.R., Dolan J.W., Molnar I., and Djordjevic, N.M., Selectivity control in reversed-phase HPLC methods development varying temperature and solvent strength to optimize separations, LC-GC, 15 (2), 136, 1997. 

Since a large majority of pharmaceutical products are amenable to reversed-phase HPLC, this is usually the method of choice. The development of reversed-phase HPLC methods is a broad subject with many research articles and books devoted to it and it is not practical to try to cover this topic in depth in this chapter. There are, however, some major points to consider when developing an HPLC method for analyzing stress test samples. 

Sultana et al. [88] developed a reversed-phase HPLC method for the simultaneous determination of omeprazole in Risek capsules. Omeprazole and the internal standard, diazepam, were separated by Shim-pack CLC-ODS (0.4 x 25 cm, 5 m) column. The mobile phase was methanol-water (80 20), pumped isocratically at ambient temperature. Analysis was run at a flow-rate of 1 ml/min at a detection wavelength of 302 nm. The method was specific and sensitive with a detection limit of 3.5 ng/ml at a signal-to-noise ratio of 4 1. The limit of quantification was set at 6.25 ng/ml. The calibration curve was linear over a concentration range of 6.25—1280 ng/ml. Precision and accuracy, demonstrated by within-day, between-day assay, and interoperator assays were lower than 10%. 

Hassib et al. developed two chromatographic procedures for the simultaneous determination of benazepril hydrochloride and hydrochlorothiazide in laboratory made mixtures, and in pharmaceutical dosage forms (Cibadrex tablets) using reversed phase HPLC and TLC methods [24]. For reversed phase HPLC, a very sensitive, rapid, and selective method was developed. The linearity ranges were 32-448 ng/ 20 pL and 40-560 ng/20 pL for benazepril hydrochloride and hydrochlorothiazide, respectively. The corresponding recoveries were 99.38 1.526 and 99.2 1.123. The minimum detection limits were 7 ng/20 pL for benazepril and 14 ng/20 pL for hydrochlorothiazide. The method could be successfully applied for the determination of laboratory made mixtures and for pharmaceutical dosage forms. The results obtained were compared with those obtained by a spectrophotometric method. 

Hasan et al. [21] developed a reversed-phase HPLC method for the determination of lomoxicam. The method is using acetonitrile phosphate buffer (pH 6) (50 50, v/v) as mobile phase at a flow rate of 1 ml/min and UV detection at 275 nm. This method is suitable as a stability indicating method for the simultaneous determination of lomoxicam in presence of its acid-induced degradates either in bulk powder or in pharmaceutical formulations. 

Attimarad [44] developed a reversed-phase HPLC method for the determination of lornoxicam in bulk and pharmaceutical preparation. Separation was performed on an Eclipse Cig column (15 cm x 4.6 mm, pm) as stationary phase and mobile phase used is methanol 0.1% formic acid in water (80 20), with a flow rate of 0.8 ml/min and UV detection at 381 nm. The method was validated for linearity, accuracy, precision, limit of detection, and limit of quantitation. Linearity, accuracy, and precision were found to be acceptable over the range 0.5-20 pg/ml. The method can be adopted for routine quality control analysis of lornoxicam. 

Counter-ions, usually small polar or ionic compounds, are routinely used to enhance the aqueous solubility and/or stability of the API. Because of their polarity, counter-ions are rarely resolved from the chromatographic solvent front in reversed-phase HPLC and have characteristically poor chromophores which makes detection difficult. The counter-ion can be omitted from the achiral method development sample set with minimal risk when this holds true. Analysis of counter-ions is normally performed using ion chromatography.9,10 This assay is separate from the reversed-phase assay performed to measure organic impurity levels. 

Fogel et. ah (53) also developed a reversed phase HPLC method for the simultaneous assay of aspirin and salicylic acid in film-coated aspirin tablets. As little as 0.1% salicylic acid (relative to aspirin) can be quantitatively determined. Using a 5-microns octadecylsilane column with water-acetonitrile-phosphoric acid (76-24 0.5) as the mobile phase enabled the chromatographic separation to be completed in 4 minutes. Due to the slow rate of decomposition of aspirin to salicylic acid in the extraction solvent, acetonitrile-methanol-phosphoric acid (92 8 0.5), the analysis of many samples was routinely performed by means of automated HPLC equipment. 

Although not a requisite for the selection of SFC as the purification technique of choice, highly lipophilic samples are preferable so as to avoid solubility problems often encountered in reverse-phase HPLC processing. It should be noted, however, that highly polar materials can be purified by SFC so long as materials can be dissolved at a level of 50m mL of methanol, along with co-solvents such as acetonitrile or dichloromethane. Further, when a synthetic chemist specifically requests that samples be returned in their non-TFA salt form, SFC is selected as it can be frequently developed in methanol/carbon dioxide or, alternatively, volatile small amine modifiers such as triethylamine. Finally, it is generally possible to achieve better resolution with an SFC rather than the HPLC method of our present setup, so in instances where a difficult separation of desired product is anticipated based on LC/MS analysis, SFC may be preferentially chosen over HPLC. 

Perucka and Oleszek have reported extraction and determination of capsaicinoids in fresh fruit of hot pepper using spectrophotometry and HPLC techniques [79]. Cooper et al. developed a reversed-phase HPLC method utilizing a conventional Cjg column to separate capsaicin, dihydrocapsaicin, and nordihydrocapsaicin present in hot peppers [121]. The isocratic mobile phase (60 40 [v/v] methanol/water at a flow rate of 1.5 mL/min) was employed and achieved the separation of these three capsaicinoids in 28 min [121]. Krajewska and Power developed a reversed-phase HPLC... 

Multifactorial Systematic Method Development and Optimization in Reversed-Phase HPLC... 

Multifactoria Systematic Method Development and Optimization in Reversed-Phase HPLC 615 DAD1 A, Sig=300,4 Ref=450,4 (E DATEN AMD1.D)... 

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