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Title: Miniaturization in the field of liquid chromatography for pharmaceutical analysis
Other Titles: Miniaturizatie van vloeistofchromatografie voor farmaceutische analyse
Authors: Hendrickx, Stijn
Issue Date: 4-Oct-2016
Abstract: During the last decades, there has been a concise trend towards miniaturization of liquid chromatography (LC) systems, with a notable increase since 2005. When only limited sample volumes are available, an increase in sensitivity can be obtained by decreasing the inner diameter of the chromatographic column. The accompanying low optimum flow rates enable a more efficient interface with the electrospray of the mass spectrometer, since the reduced flow rate will result in smaller electrospray droplets. An additional advantage of miniaturization is the reduction of often hazardous organic mobile phase solvents and amounts of stationary phase material needed. However, due to the miniaturization of the i.d. of the column, the relative importance of extra-column band broadening is significantly increased and measures need to be taken to minimize extra column void volumes.
The main aim of this research work was to demonstrate the applicability of miniaturized LC systems for bioanalytically and pharmaceutically relevant separations. Therefore, miniaturized LC setups were utilized to tackle a number of real-life applications in the field of pharmaceutical analysis. Emphasis was given to the benefits and drawbacks of the use of miniaturized LC for the specific characteristics of the research projects.
In a first application, a highly efficient capillary LC method coupled to time-of-flight mass spectrometry detection was developed to separate and identify as many steviol glycosides as possible in a complex plant extract of Stevia rebaudiana Bertoni. Extracts of Stevia are increasingly being used in the food industry as natural sweeteners, because of their sweet taste and low-caloric characteristics. Up till now, 21 different steviol glycosides, the compounds responsible for the sweet taste of Stevia, have been described in literature. Previous research has moreover indicated the existence of a number of unidentified steviol glycosides that have not yet been reported. It has been stated that Stevia extracts can suffer from a bitter aftertaste. It was therefore desirable to try and identify as many steviol glycosides as possible in a commercial Stevia extract and to develop a method for structural elucidation of unknown steviol glycosides. This could present as a first step in the identification of the molecules responsible for this bad aftertaste. A capillary LC method was developed and subsequently coupled to a time-of-flight mass spectrometer. Using a set of steviol glycoside standards, it was shown that the fragmentation behavior at different fragmentation voltages could be used for identification and structural elucidation of unknown steviol glycosides. A general strategy to process the vast amount of LC-MS data and to correct for small retention time shifts was developed.

Even though the data suffered from retention time shifts and low repeatability of the fragmentation, the potential of the capillary LC-MS method was demonstrated by tentatively identifying a total number of 32 steviol glycosides based on their parent mass, the formation of a known steviol glycoside fragment and their distinct retention time. This is significantly more than the 21 currently described steviol glycosides. The possibility to use a miniaturized column in the first dimension of an online heart-cut 2D-LC method in order to improve the peak capacity and ionization efficiency was also briefly explored, but the setup suffered from sensitivity and peak broadening issues.
The second application consisted of the development and validation of a specific and sensitive capillary LC-UV method for the simultaneous separation and quantification of two antipsychotics and their FMO mediated N-oxides in rat brain microdialysates. Since microdialysis samples generally only yield small sample volumes with low concentrated analytes, capillary LC in combination with a high injection volume was chosen in order to maximize sensitivity. To eliminate UV interferences from the sample, a MEPS sample clean up procedure was developed. A capillary LC-UV method was successfully developed and validated in terms of linearity, sensitivity, recovery, precision and stability. By focusing the analytes at the top of the column at the start of the method, an injection volume as high as 20 µL could be used without the occurrence of severe peak broadening or loss of resolution. As a result, an LLOQ of 0.5 nM could be obtained for all compounds with good MEPS recoveries and acceptable precision. The applicability of the method was demonstrated by analyzing a number of microdialysate samples from rats after i.p. administration of OLA, CPZ or both. The capillary LC-UV method in combination with MEPS sample treatment provides a simple, sensitive method to quantify all compounds of interest in an acceptable analysis time and can be applied to routine therapeutic monitoring and pharmacokinetic studies of olanzapine, chlorpromazine and their respective N-oxides.
Both applications demonstrate the potential of miniaturized LC for pharmaceutical and bioanalytical applications. A number of disadvantages, however, still obstruct the implementation of miniaturized LC in routine applications. The miniaturized LC system is far less robust than the traditional-bore LC in terms of extra-column variance. Proneness to clogging and strenuous leakage detection can be very difficult and time-consuming to solve and a source of irritation for end-users. And finally, miniaturized LC systems and consumables are considerably more expensive than their normal-bore counterparts. Even though improvements that can solve a number of these issues are currently being developed, it remains to be seen whether miniaturized LC can acquire a prominent place in biomedical and pharmaceutical analysis in the near future.
Table of Contents: Curriculum Vitae i
Acknowledgements iii
Publications v
List of abbreviations and symbols xiii


Chapter 1: Introduction……………………………………………………………………………….…………………………...……1

1.1. Trends and challenges in pharmaceutical analysis 3
1.1.1. Modern pharmaceutical analysis 3
1.1.2. Trends in modern pharmaceutical analysis 4
1.1.2.1. Hyphenation of analytical techniques 4
1.1.2.2. High-throughput analysis 4
1.1.2.3. Miniaturization and nanotechnology 5
1.2. Principles of high performance liquid chromatography (HPLC) 6
1.2.1. History of HPLC 6
1.2.2. Separation mechanisms 7
1.2.2.1. Separation mechanisms based on difference in polarity 7
1.2.2.2. Separation mechanisms based on charge 8
1.2.2.3. Separation mechanisms based on molecular size 9
1.2.3. Detectors 9
1.2.3.1. UV-VIS detection 9
1.2.3.2. Mass spectrometry 10
1.2.4. Basic theory in LC 12
1.2.4.1. Retention time and retention factor 13
1.2.4.2. Band broadening and plate count 13
1.2.4.3. Band broadening and van Deemter equation 14
1.2.4.4. Selectivity and resolution 15
1.3. Miniaturization of liquid chromatography 16
1.3.1. Advantages of miniaturized LC 17
1.3.1.1. Increase in sensitivity 17
1.3.1.2. Additional advantages 17
1.3.2. Analytical instrumentation of nano-LC systems 18
1.3.2.1. Extra column band broadening 18
1.3.2.2. Detectors 18
1.3.3. Applications of miniaturized LC 19
1.4. Sample preparation techniques 21
1.4.1. The need for sample preparation 21
1.4.2.1. Sample treatment techniques in HPLC 23
1.4.2.1. Protein precipitation 23
1.4.2.2. Liquid-liquid extraction 23
1.4.2.3. Solid phase extraction 24
1.5. References 26


Chapter 2: Objective…………………………………………………………………………………………………….……….….….33


Chapter 3: Development of a capillary LC-MS method for the identification of novel steviol glycosides in Stevia rebaudiana Bertoni……………….…………………………………………………………………………………...….37

ABSTRACT 39
3.1. Introduction 40
3.2. Material and methods 44
3.2.1. Chemicals and sample stock solutions 44
3.2.2. Instrumentation 45
3.2.3. Comparison of columns and optimization of the method using the LSS model 46
3.2.3.1. Theory 46
3.2.3.2. Practical application 47
3.2.4. Structure elucidation by LC-MS 47
3.2.5. Heart-cut 2D-LC 48
3.3. Results and discussion 49
3.3.1. Determination of fragmentation patterns of steviol glycoside standards 49
3.3.2. Choice of column and method development using the LSS model 53
3.3.3. Hyphenation of cap-LC method to MS and identification strategy for steviol glycosides 55
3.3.4. Re-optimization of chromatographic conditions for coupling to MS 61
3.3.5. Re-optimized separation coupled to MS 63
3.3.6. Heart-cut two-dimensional liquid chromatography 65
3.4. Conclusion 68
3.5. References 69
3.6. Appendix: Results of the quantification of retention time shifts during first coupling to MS 72


Chapter 4: A sensitive capillary LC-UV method for the simultaneous analysis of olanzapine, chlorpromazine and their FMO-mediated N-oxidation products in rat brain microdialysates……….75

ABSTRACT 77
4.1. Introduction 78
4.2. Material and methods 85
4.2.1. Chemicals and reagents 85
4.2.2. Preparation of solutions 85
4.2.3. Instrumentation and chromatographic conditions 85
4.2.4. MEPS procedure 86
4.2.5. Microdialysis procedure 87
4.2.6. Method validation 88
4.2.6.1. MEPS recovery, precision and accuracy 88
4.2.6.2. Linearity, lower limit of quantification 88
4.2.6.3. Specificity and selectivity 88
4.2.6.4. Stability 89
4.3. Results and discussion 89
4.3.1. Optimization of chromatographic conditions 89
4.3.2. MEPS optimization 93
4.3.3. Method validation 94
4.3.3.1. Specificity and selectivity 94
4.3.3.2. Linearity and LLOQ 95
4.3.3.3. MEPS recovery, accuracy and precision 95
4.3.3.4. Stability 97
4.3.4. Method application 97
4.4. Conclusion 99
4.5. References 100


Chapter 5: General discussion and conclusion…….……………………………………….……….……………….…...105

5.1. Implementation of miniaturized LC 107
5.2. Stevia Rebaudiana Bertoni 110
5.3. Quantification of antipsychotics and their N-oxides 113
5.4. Drawbacks of miniaturized LC and future outlook 115
5.5. References 116


Chapter 6: Summary / samenvatting…………………..…….……………………………………….……………………….119

6.1. Summary 121
6.2. Samenvatting 123
Publication status: accepted
KU Leuven publication type: TH
Appears in Collections:Pharmaceutical Analysis

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