Method development for HPLC

Figure 25-28 Isocratic method development for HPLC, using solvent composition, % B. and temperature, T, as independent variables. % B and T are each varied between selected low and high values. From the appearance of chromatograms resulting from conditions A-D, we can select intermediate conditions to improve the separation.

Sample handling is a very important part of the method development for HPLC determination of phenolic acids in natural plants. Because of the great variability of phenolic acids (different polarity, acidity, number of hydroxyl groups, and aromatic rings), the various concentration levels of individual analytes, and the very complex natural matrix with many interfering components, the choice of the technique for their isolation and quantification differs from one described HPLC assay to the next. In some cases, only a one-step extraction and simple clean-up procedure are sufficient before the HPLC analysis, but the most often described HPLC assays include two or more steps of sample preparation, especially in the case of fruits and vegetable samples. It is obvious that each step contributes, on one hand, to the higher sensitivity and selectivity, but, on the other hand, it could increase the number of errors and decrease the recovery of the method. 

Xu et al. [43] published a report on a process for rapid HPLC-MS/MS method development in a drug discovery setting. In this report, the authors provided a three-step process for rapid bioanalytical method development for HPLC-MS/MS methods suitable for drug discovery PK applications. Figure 1.10 shows the flowchart... 

Sample handling is a very important part of the method development for HPLC determination of phenolic acids in... 

Scotter, M.J., Castle, L., and Roberts, D., Method development and HPLC analysis of retail foods and beverages for copper chlorophyll (E 141 [i]) and chlorophyllin (E 141[ii]) food colouring materials, FoodAddit. Contam., 22, 1163, 2005. 

Procedures used vary from trial-and-error methods to more sophisticated approaches including the window diagram, the simplex method, the PRISMA method, chemometric method, or computer-assisted methods. Many of these procedures were originally developed for HPLC and were apphed to TLC with appropriate changes in methodology. In the majority of the procedures, a set of solvents is selected as components of the mobile phase and one of the mentioned procedures is then used to optimize their relative proportions. Chemometric methods make possible to choose the minimum number of chromatographic systems needed to perform the best separation. 

One issue related to supporting a metabolic stability assay with HPLC/MS/MS is the need to set up an MS/MS method for each compound. While it may only take 10 min to infuse a compound solution and find the corresponding precursor and product ions (along with minimal optimization of the collision energy), the processes of MS/MS development would require 4 hr per day if one wanted to assay 25 compounds per day. MS vendors have responded to this need by providing software tools that can perform the MS/MS method development step in an automated fashion. Chovan et al.68 described the use of the Automaton software package supplied by PE Sciex (Toronto, Canada) as a tool for the automated MS/MS method development for a series of compounds. The Automaton software was able to select the correct precursor and product ions for the various compounds and optimize the collision energy used for the MS/MS assays of each compound. They found that the Automaton software provided similar sensitivity to methods that would have been developed by manual MS/MS procedures. Chovan et al. also reported that the MS/MS method development for 25 compounds could be performed in about an hour with the Automaton software and required minimal human intervention. 

FIGURE 21 (a) An isocratic reversed-phase HPLC method developed for assay, with which 15%... 

Jimidar, M., and De Smet, M. (2007). HPLC method development in late phase pharmaceutical development. In HPLC Method Development For Pharmaceuticals (S. Ahuja, and H. Rasmussen, Eds), Vol. 8 of Separation Science and Technology, pp. 373—405, Academic Press, Elsevier, London, Chapter 13. 

Fig. 13.5 A schematic diagram showing the stepwise procedure for rapid method development of HPLC-MS/MS methods for discovery PK assays. Adapted from [10], with permission from the American Chemical Society.

Analytical Method Development for TRIS. The detection of brominated compounds of very low volatility such as TRIS posed special analytical problems. Since TRIS has no recognizable chromophore, the detection systems which are commonly used with high performance liquid chromatography (hplc), such as refractive index or short wavelength (<220 nm) uv detectors, are too non-specific to be of much practical use for the analysis of environmental samples. Furthermore, the sensitivities available with these detection methods are generally inadequate. 

Common HPLC detectors lack the sensitivity and selectivity to determine the low concentrations of the endogenous B,2 vitamers in foods. As a result, vitamin Bl2 method development for food samples has concentrated on ligand binding and microbiological assays no reviews of HPLC methods have been published. 

The most recent paper on this topic has been published by Lu and Huang (213). The method consists of an online enrichment of the aromatic amines on a carboxymethyl-bonded silica precolumn and an HPLC-UV (at 254 nm) analysis. The mobile phase, ACN-acetate buffer (pH 4.66) (40 60, v/v), was used to desorb the analytes and for the subsequent separation. The method was applied to the determination of several compounds (4-aminoazobenzene (4-AAB), benzidine (Bz), 3,3 -methylbenzidine (DMBz), 4-aminobiphenyl (4-ABP), 3,3 -dichlorobenzi-dine (DCBz), and 2-naphthylamine (2-NA) together with some substituted naphthalens and phenols) in aqueous solution of four food dyes Direct Blue 6, Amaranth, Sunset Yellow FCF, and D C Orange No. 4. Detection limits ranged between 0.6 and 1.6 fig/g. Most part of the methods developed for this kind of determination are reported in Table 3. 

Because of this concern, a variety of methods have been developed for the determination of NOC in foods. These include thin-layer chromatographic (TLC) (11,12), gas chromatographic (GC) (13-15), GC-mass spectrometric (GC-MS) (15-17), and high-performance liquid chromatographic (HPLC) (18-20) techniques. The purpose of this article is to review various HPLC methods developed for this purpose. Unfortunately, however, only limited advances have been made in this area, mainly because of the lack of sensitive and specific detectors. Most published methods for NOC reported to date are GC-based techniques. Therefore, this review will be a brief one and will emphasize the most recent HPLC developments. 

Benzoic and sorbic acids are now normally assayed using HPLC. As discussed in the section on the analysis of sweeteners, some of the HPLC methods developed for soft drinks actually allow the separation of both sweeteners and preservatives in one ran, for example, Williams (1986), Hagenauer-Hener et al., (1990) and the EU method for sweeteners (Anon, 1999a), although the preservatives were not included in the collaborative trial of the method. The separation of benzoic and sorbic acids can sometimes be difficult and care should be taken that the system will actually resolve these two preservatives if they are present otherwise spurious results can be obtained. The pH of the solvent is a critical feature that allows the separ ation of these two preservatives. 

A word of warning if you are planning to do automated methods development. The HPLC system you buy will generally dictate the type of methods development software that is available to you this is not an add-on procedure that you can purchase at a later time, except from the manufacturer of your system computer. If you are contemplating a need for this technique, I would recommend strongly that you talk to other people who are already successfully doing this type of work and find out their HPLC system source and buy from the same place. 

HPLC method development for the fixed combination tablet formulation became more challenging in the presence of the excipients used in the tablet formulations. HPMC and pregelatinized starch in the sustained release portion of the bilayer tablet caused extraction difficulties and interferences in the HPLC chromatogram. 

The authors would like to thank Vincent Bobin for the solubility data for Compound A. The authors would also like to thank the following individuals for their work on Compound B described in this chapter Daniel Gierer for manufacture of the placebo tablets Amy Orce for her work on the extraneous syringe peak Thomas Sharp, George Horan, and Ronald Morris for their work on impurity identification and Cheryl Kirkman, Heidi O Donnell, Britt-Marie Otano, Doreathea Roberts, J. Sean Space, and Gregory Steeno for their work on the small volume dissolution method. In addition, the authors would like to thank Amanda Deal and Kelly Field for their work on the HPLC purity method development for the fixed combination tablet. 

Plasma carboxypeptidase N degrades and therefore inactivates bradykinin. This activity may have a role in the regulation of inflammatory peptides. The HPLC method developed for its assay uses the dipeptide hippuryllysine (Hip-Lys) as the substrate and measures activity by measuring the release of hip-puric acid. 

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. 

Creatine kinase catalyzes the reversible reaction whereby ADP + phosphocre-atine form ATP + creatine. One HPLC method developed for this activity involved the direct determination of the ATP formed. 

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... 

Knowledge of pKa for the target analyte and related impurities is particularly useful for commencement of method development of HPLC methods for key raw materials, reaction monitoring, and active pharmaceutical ingredients. This practice leads to faster method development, rugged methods, and an accurate description of the analyte retention as a function of pH at varying organic compositions. Relationship of the analyte retention as function of mobile-phase pH (spH) is very useful to determine the pK of the particular... 

A well-defined method development plan with clear aim of analysis is critical to the success for fast and effective method development. The general approach for the method development for the separation of pharmaceutical compounds was discussed, emphasizing that modifications in the mobile phase (organic and pH) play a dramatic role on the separation selectivity. The knowledge of the Ka of the primary compound is of utmost importance prior to the commencement of HPLC method development. Moreover, pH screening experiments can help to discern the ionizable nature of the other impurities (i.e., synthetic by-products, metabolites, degradation products, etc.) in the mixture. 

S. V. Galushko, Software for method development in HPLC, GIT Spezial Chro-matogr. 16 (1996), 88-93. 

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