Reversed-phase HPLC pharmaceuticals

Gagliardi, L., De Orsi, D Manna, L, Tonelli, D. Simultaneous determination of antioxidants and preservatives in cosmetics and pharmaceutical preparations by reversed phase HPLC./. Liquid Chromatogr. Rel. Technol. 1997, 20, 1979-1808. 

Amin, M., Simultaneous determination of prostaglandins (PG) E2, A2 and B2 and stability studies of PGE2 in pharmaceutical preparations by ion-pair reversed phase HPLC, Pharm. Acta. Helv., 64, 45, 1989. 

Note that our primary focus is on reversed-phase HPLC (RPLC) since it is the predominant mode for pharmaceutical analysis. Many of these concepts, however, are applicable to other modes of HPLC such as ion-exchange, adsorption, and gel-permeation chromatography. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

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. 

El-Sherif et al. [79] developed and validated a reversed-phase HPLC method for the quantitative determination of omeprazole and two other proton pump inhibitors in the presence of their acid-induced degradation products. The drugs were monitored at 280 nm using Nova-Pak Ci8 column and mobile phase consisting of 0.05 M potassium dihydrogen phosphate-methanol-acetonitrile (5 3 2). Linearity range for omeprazole was 2-36 fig/ml. The recovery of omeprazole was 100.50 0.8%, and the minimum detection was 0.54 /zg/ml. The method was applied to the determination of pure, laboratory prepared mixtures, and pharmaceutical dosage forms. The results were compared with the official USP method for omeprazole. 

K. R. Bagon, The assay of antibiotics in pharmaceutical preparations using reversed-phase HPLC, HRC CC. J. High Resol. Chromatogr. Chromatogr. Commun., 2 211 (1979). 

I. M. Jalal and S. I. Sa sa, Simultaneous determination of dextro-propoxyphene napsylate, caffeine, aspirin and salicylic acid in pharmaceutical preparations by reversed-phase HPLC, Talanta, 37 1015 (1984). 

For pharmaceutical compounds, LC-MS has found extremely wide acceptance due to the low-level detection that can be achieved, in addition to the selectivity and specificity that are attained by using HPLC in conjunction with MS detection. LC-MS is also convenient because of its compatibility with reversed-phase HPLC mobile phases. Volatile mobile phase additives such as trifluoroacetic acid, formic acid, and ammonium hydroxide are very common and can be utilized not only to aid in the chromatographic separation but also to influence the ionization state of the molecule (i.e., acid modifiers to protonate [M + H]+1, and basic modifiers to deprotonate [M — H] ). This requirement may require a modification of the potency method if phosphate was utilized however, it is not viewed as a major drawback. 

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. 

Reversed-phase HPLC will find increased application in the analysis of purine antimetabolites and nucleoside antibiotics, both in the chemical laboratory for monitoring serum levels in chemotherapeutic treatment and in quality control in the pharmaceutical industry. In addition, RPLC will be used as a clinical diagnostic tool and aid the clinician in the detection of disease, in confirming a diagnosis, and in monitoring the causes of disease or effectiveness of therapy. 

Reversed-phase HPLC is the dominant method used for the majority of pharmaceutical applications (>95%). Normal-phase chromatography may be required for separations that are not compatible with reversed-phase mode. 

D. Louden, A. Handley, S. Taylor, I. Sinclair, E. Lenz, and I. D. Wilson, High temperature reversed-phase HPLC using deuterium oxide as a mobile phase for the separation of model pharmaceuticals with multiple on-line spectroscopic analysis (UV, IR, IH-NMR and MS), Analyst 126 (2001), 1625-1629. 

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. 

By examining this brief history of the development of instrumentation and methods, it should be clear what parameters define the instrument of choice for analysis of pharmaceutically important molecules. Such an instrument is one capable of performing LC-MS, typically using a reverse-phase HPLC separation and one of the API techniques. MS-MS capability is desirable—using an ion trap, a triple quadrupole, or other tandem mass analyzer instrument. Accurate mass measurement capability is also desirable. These attributes make up a prioritized list of capabilities for the ideal full-purpose instrument to be used in a pharmaceutical research environment. As one progresses through this list, the expense of the instrument increases, the sophistication of the instrument increases, and the intellectual and technological commitment required to do these experiments increases. 

Unstable compounds are problematic. A sample purified in the laboratory might have a short shelf-life and poor performance as a standard. Compounds altered by assays are also inconvenient. For example, substituted benzylic alcohols can dehydrate under acidic HPLC conditions, or carboxylic esters can hydrolyze in aqueous mobile phase. An impurity isolated from an active pharmaceutical ingredient as an organic salt of an organic compound poses two problems at once. The analyst must account for both the acid and the base. In the case of a toluenesulfonic acid salt of an aliphatic amine, two different methods of detection might be needed. The toluenesulfonic acid in a reverse-phase HPLC assay can by monitored by UV light, but the aliphatic amine, with no chromophore, must be measured by a different technique. 

Santos-Montes, A. Gasco-L6pez, A.I. Izquierdo-Hornillos, R. Simultaneous determination of dexamethasone and betamethasone in pharmaceuticals by reversed-phase HPLC. Chromatographia, 1994, 39, 539-542 [simultaneous dexamethasone methylprednisolone (IS) tablets column temp 30 LOD 6 ng/mL non-interfering corticosterone, cortisone, deflazacort, fludrocortisone, fludrocortisone acetate, hydrocortisone, hydroxyprogesterone, methylprednisolone, prednisolone, prednisone, triamcinolone interfering triamcinolone acetonide]... 

Spangler, M. Isocratic reversed phase HPLC analysis of a pharmaceutical cream. Supelco Reporter, 1994, 13(2), 12-13... 

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