Assay methods, HPLC

The case stndy of the SP development of an HPLC assay method for an OTC prodnct derived from natural materials illustrates the importance of optimizing both the extraction and filtration conditions to fully recover all the labile active components in the formulation. The HPLC gradient assay method, which separates all active ingredients from other... 

Other HPLC assay methods reported in the literature [130-143] are shown in Table 8. 

Motevalian et al. [62] developed a rapid, simple, and sensitive HPLC assay method for the simultaneous determination of omeprazole and its major metabolites in human plasma using a solid-phase extraction procedure. Eluent (50 /d) was injected on a /rBondapak Ci8 reversed-phase column (4.6 mm x 250 mm, 10 /un). The mobile phase consisted of 0.05 M phosphate buffer (pH 7.5) and acetonitrile (75 25) at a flow-rate of 0.8 ml/min. UV detection was at 302 nm. Mean recovery was greater than 96% and the analytical responses were linear over the omeprazole concentration range of 50-2000 ng/ml. The minimum detection limits were 10, 10, and 15 ng/ml for omeprazole, omeprazole sulfone, and hydroxyomeprazole, respectively. The method was used to determine the plasma concentration of the respective analytes in four healthy volunteers after an oral dose of 40 mg of omeprazole. 

Rezk et al. [74] developed and validated a reversed-phase HPLC assay method for the simultaneous quantitative determination of omeprazole and its three metabolites in human plasma. The method provides excellent chromatographic resolution and peak shape for the four components and the internal standard within a 17-min run time. The simple extraction method results in a clean baseline and relatively high extraction efficiency. The method was validated over the range of 2-2000 ng/ml. The resolution and analysis for the four analytes omeprazole, hydroxyome-prazole, omeprazole sulfone, and omeprazole sulfide and the internal standard utilized a Zorbax C18 (15 cm x 3 mm, 5 /im) with a Zorbax C18 (12.5 cm x 4.6 mm) guard column. The mobile phase consisted of two components. Mobile phase A was 22 mM phosphate monobasic, adjusted to a pH of 6 with diluted sodium hydroxide. This solution was filtered through a 0.45-/im membrane filter, then mixed as 900 ml buffer to 100 ml methanol. Mobile phase B was composed of 100 ml of the phosphate buffer as mobile phase A, mixed with 800 ml of acetonitrile, 100 ml of methanol, and 100 /A of trifluoroacetic acid with an initial flow-rate of 0.55 ml/min and detection at 302 nm. 

A final point to be considered in the use of HPLC as an assay procedure is the enzyme itself. Will the activity be a pure enzyme Will it be part of a rather crude cell-free extract Or will it be present in a fermentation broth In the latter two cases, the presence of contaminating activities must be considered. While as mentioned above, these activities, by affecting the recovery of the product or even by affecting substrate levels during the course of the reaction, could easily cause problems with other assay procedures, they are not a problem for the HPLC assay method. Thus, HPLC should be considered first when activity in some crude extracts is to be assayed. 

Modification of Reaction Conditions for the HPLC Assay Method... 

The HPLC assay method is particularly useful when it is necessary to obtain initial rate data for a study of an enzymatic activity. Optimal assay conditions for the HPLC must be established first. Usually, the optimization process involves the determination of several variables, such as the optimal substrate concentration, pH, temperature, and enzyme concentration. It is assumed that the reader is familiar with the problems associated with assay conditions such as pH, buffer, and temperature. This chapter discusses only factors that might present problems for the HPLC assay method. For additional information, see the works cited in the General References. 

The significance of obtaining rate data for the study of enzymes has been discussed elsewhere, and the reader is referred to the General References for additional information. Although usually relevant to the in-depth study of the mechanism of an enzyme reaction, such concerns are beyond the scope of the present discussion. Of concern in this text are the problems associated with obtaining initial rate data with the HPLC assay method. 

For the two-substrate-two-product reactions, the BiBi reactions in Cle-land s nomenclature, the HPLC assay method is particularly useful. Its application, however, will require the development of methods for the separation of the two substrates and the two products. Initial rate data generated by the... 

The application of the HPLC assay method to studies on reaction mechanisms has been limited, and the reader is referred to the work of Sloan (1984). Sloan and his colleagues studied the formation of IMP or GMP (and pyrophosphate) from the substrates phosphoribosylpyrophosphate (PRPP) and either hypoxanthine or guanine. These reactions, catalyzed by hypoxan-thine/guanine phosphoribosyltransferase (GHPRTase), were studied by HPLC after a method was developed to separate all the reactants and products simultaneously. 

While such a study might have been carried out with conventional methods, the use of HPLC facilitated the work considerably by allowing all reactants and products to be measured in one analysis. Clearly, the HPLC assay method should be considered when the kinetics of a multisubstrate enzyme reaction are to be studied. 

Determination of kinetic mechanisms by the HPLC assay method... 

The general reference section contains additional citations that are relevant to the enzymes described, to provide readers with a more extensive survey of the HPLC assay methods that have been developed for these activities. 

The HPLC assay method developed for this activity involves the separation of the three compounds NANA, CTP, and CMP-NANA. However, at the wavelength used for detection, only the last two are detected. 

Since thioadenosine is readily separated from adenosine by HPLC, the use of this analog together with the HPLC assay method allowed the site of cleavage to be established. As shown in Figure 9.93A, an analysis of the incubation mixture during the course of the reaction revealed the formation of thioadenosine. No adenosine was detected. These findings supported the conclusion that the site of cleavage was the bond between the sulfur and the phosphate. 

Measurement of the activity of this enzyme by the HPLC assay method requires separation of the hypoxanthine and the IMP, which can be easily accomplished by several methods, including reversed-phase or ion-exchange HPLC. Jahngen and Rossomando (1984) used a reversed-phase Ci8 column with a mobile phase of 10 mM potassium phosphate (pH 5.6), and 10% methanol. Detection was at 254 nm, and the separation obtained is shown in Figure 9.98. 

Figure 9.111 Adenylate kinase activity measured by the HPLC assay method. The assay mixture contained 200 fiM ATP, 20 fiM pH]AMP (approximately 120,000 cpm), 50 mM Tris-HCl (pH 7.4), and 32 fig of protein of the enzyme from Dictyostelium discoideum. Samples (20 juL) were injected and the radioactivity monitored continuously with a Berthold LB 503 detector using PICO-Fluor 30 scintillant. Absorbance was measured at 254 nm. Three representative time points were shown (A) 1 minute (B) 30 minutes, and (C) 60 minutes after initiation of the reaction. (From Rosso-mando, 1987.)... 

Since the HPLC assay method can measure several related components simultaneously, it is possible to monitor the activity of a single enzyme among multiple catalysts. In addition, because many of the enzymes that process related compounds represent a pathway, the HPLC method makes it possible to study the flow of a compound through such a multienzyme complex. This chapter explores the application of the HPLC assay method to... 

In one type of study, the HPLC assay method has been applied to the question of assaying the activity of two enzymes that can use the same substrate but form different products. In another, the HPLC method was applied to the case of related two different enzymes that can use the same substrate and form the same product. It was also applied to the case a single enzyme that can use either of two substrates to form two different products. In a separate application, the so-called reconstitution approach, a multienzyme pathway is reconstructed by the addition of enzymes one after the other to form a multienzyme complex and to thereby reconstitute a naturally occurring multienzyme complex. And finally, this approach allows us to use the HPLC method to study an intact, naturally occurring multienzyme system. 

The enzyme 5 -nucleotidase dephosphorylates IMP to inosine and P. Thus, since this reaction represents a possible fate for the IMP formed by the transferase (Fig. 10.7), reconstitution studies were undertaken with the nucleotidase. These studies were carried out using the HPLC assay method developed for the HGPRTase activity. A reaction mixture was prepared that contained hypoxanthine and PRibPP as substrates. The reaction was started by the addition of purified HGPRTase enzyme. Samples were removed and were analyzed by HPLC. The chromatographic profiles obtained at 0,10, 20, and... 

Prabhakar et al. [56] developed and validated six HPLC assay methods which permitted resolution of tailing problems for the separation of phenothiazine bioactive components and preservatives in liquid pharmaceutical formulations by using cyano-, C-8, C-18, and cation-exchange phases. The mass spectrometry (MS) of phenothiazines in biological samples is important in forensic analysis. However, since most phenothiazines having long piper-azinyl side chains are thermolabile, the phenothiazines are not suitable for conventional gas chromatography (GC)/MS analysis. Therefore, a system of capillary HPLC/fast atom bombardment-mass spectrometry (FAB-MS) was... 

LeBel, M. Ericson, J.F. Pitkin, D.H. Improved high-performance liquid chromatographic (HPLC) assay method for ceftizoxime. J.Liq.Chromatogr., 1984, 7, 961—968 [simultaneous ceftizoxime cefotaxime is IS]... 

Hou S, Hindle M, Byron P (2001) A stability-indicating HPLC assay method for budesonide. J Pharm Biomed Anal 24 371-380. 

Based on the known amount of analyte spiked into the sample, % recovery is calculated and compared to pre-set acceptance criteria. The acceptance criteria will depend on the ruggedness of the method, for a typical small molecule HPLC assay method it is usually set at a mean of 98-102% or 97-103% of theoretical, with individual values allowed to be a bit wider. An example of results obtained from a recovery study for XYZ Tablets by one analyst is presented in Table 8.1. For a large molecule assay method, the acceptance criteria would be set wider consistent with the difficulty of the method. Acceptance criteria for impurity methods typically widen as the concentration decreases, thus a 0.1% level, 20% (80-120% recovery) may be set where as at a 1 % level the criteria may be set at 5% for the mean. 

RSD of 2% or less for a HPLC assay method for a small molecule drug product or API. For impurity measurements, the %RSD will increase as the spiked level decreases. Typical acceptance criteria at 0.1% levels are 10-25% RSDs, whereas at a 1% level, %RSD criteria are set at 3-5%. An example of accuracy and precision results obtained from a recovery study for Degradation Product A from XYZ Tablets by one analyst is presented in Table 8.2. Another method for measuring repeatability is to analyze a homogenous sample multiple times, for example 6 x samples at 100% of test concentration and then determine the %RSD. 

HPLC assay method, 50, 80, 100, 120, and 150% of target concentration are prepared and analyzed. Typical parameters reported from a linear regression analysis are the correlation coefficient (r) with an acceptance criteria typically greater than... 

When using the HPLC assay method, contaminations from the protein boimd iodine do not interfere with the determination of the serum inorganic iodide (SII), making it the method of choice for detection in the serum. Although the clinical relevance of the measurement of SII is limited, it allows calculation of the absolute iodine uptake, which has a great value in certain pathophysiological studies (Rendl et al., 1998). 

M. Bakshi and S. Singh, HPLC and LC-MS studies on stress degradation behaviour of tinidazole and development of a validated specific stability-indicating HPLC assay method, Journal of Pharmaceutical and Biomedical Analysis, vol. 34, no. 1, pp. 11-18, 2004. 

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