UHPLC Method Development

HPLC method development principles can be applied to UHPLC method development, although detailed procedures may differ. In addition, many existing HPLC methods used in the pharmaceutical industry can be converted to UHPLC methods. In practice, a UHPLC method may need to be converted to HPLC when a UHPLC system is not available. 

This chapter provides an overview of the UHPLC method development process and the conversion process of an HPLC method to UHPLC or vice versa. It mainly focuses on analytical reversed phase UHPLC method development of small molecules. A general process and detailed steps are discussed as well as practical... 

Figure 1.1 General process of UHPLC method development.

In addition, outsourcing becomes more and more common to reduce the cost of drug development in the pharmaceutical industry. UHPLC methods developed in a company with UHPLC capability need to be transferred to contract research organizations (CROs), which may not have UHPLC capability. 

Another potential benefit of UHPLC is its capability of solving the most challenging separation tasks in pharmaceutical analysis. Figure 9.4 shows a UPLC method developed to analyze pharmaceutical formulations used to treat the common cold. Cold products often contain multiple active ingredients to treat different symptoms and can contain decongestants, antihistamines, pain relievers, cough suppressants, expectorants, and numerous excipients of various polarities. The analysis of a total of 20 components was achieved within 10 min. 

UHPLC (600 to 1000 bar) Significant runtime reduction for ultra-fast separation minimal solvent consumption Five-fold increase in speed for SIM Significantly higher efficiency for most complex separations Higher mass sensitivity Rapid method development... 

It is not trivial to build pumps for use at 1000 bar which fulfill the common requirements such as pulse-free flow which is independent of the pressure drop. Nevertheless it is possible the demand for high peak capacity (e.g. needed in proteomics) or for fast method development in industry led to the appearance of such instruments on the market. The technique is called ultra high pressure hquid chromatography (UHPLC). 

An overview of several recent applications of UHPLC-MS(/MS) methods for the multi-residue analysis of pesticides in food products has been presented. In order to cope with the necessity of high throughput and fast analysis of pesticide residues in food, all aspects of analytical method development, that is, sample extraction and clean-up, chromatographic analysis, and quantitation and confirmation aspects, must be taken into account. 

In the manuscript that we are describing in depth in this chapter [73], repeatability, reproducibility, and accuracy were checked, and the calibration curves were established, allowing the calculation of the detection and quantification limits. RSD values for repeatability were lower than 7.01% accuracy values oscillated between 97.2% and 102.0% and limits of detection were low, ranging from 1.64 to 730.54 ppb (negative polarity) and from 0.51 to 310.23 ppb (positive polarity). Neither matrix effect nor ion suppression were detected. The authors proved that the method developed for UHPLC-UV-ESI-TOF MS was a very valuable tool, because it was able to determine a wide number of metabolites in a single run and it was reliable from an analytical point of view. For this reason, it was also applied for the quantitative analysis of the 26 samples under study. 

In order to obtain reliable results for quantification purposes, the quality parameters of the methods developed need to be studied. Only two developed UHPLC-MS/ MS methods have been validated in the literature. One study was validated by using the National Institnte of Standards and Technology (NIST) Certified Reference Material (CRM) [18], and the second by spiking the extraction solution with different concentrations of procyanidin and alkaloid [29]. The LOQs obtained for catechin and epicatechin were 11.2 and 65.5 pg/g of chocolate when the NIST CRM was nsed [18], On the other hand, when the extraction solution was spiked with the target analytes standards, the detection limits (LODs), and LOQs of the procyanidins (catechin, epicatechin, and the dimer Bj) were between 7 and 30 pg/L and 10 and 90 pg/L, respectively, and for the caffeine and teobromine alkaloids, these values were below 30 and 100 pg/L, respectively [26]. 

Ultra high performance liquid chromatography (UHPLC) or ultra performance liquid chromatography (UPLC) is a quite novel technique, but it is one of the most popular nowadays, due to the fact that it is much more rapid than conventional LC. UHPLC systems can work at very high pressures and are compatible with stationary phases with particle sizes of around two micrometers or even smaller. Thus, the development of UHPLC methods [69,77, 80] or the conversion of HPLC to UHPLC methods is a current trend, due mainly to the speed of analysis and low solvent use. 

Kovalczuk et al. [81] developed a high-throughput method for multiresidue pesticide analysis in food matrices based on ultra high-performance liquid chromatography (UHPLC) with coupling to tandem mass spectrometry. Separation of 17 polar pesticides was achieved in under 8 min (see Figure 13.5). The authors compared UHPLC and conventional LC approaches, and they observed higher separation efficiencies with the LC method. Faster separations and better limits of quantitation were possible with the UHPLC method, however. A different approach for multiresidue... 

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