CHAPTER 3.1 BIOANALYSIS OF RIVASTIGMINE

Transcription

1 CHAPTER 3.1 BIOAALYSIS OF RIVASTIGMIE 51

2 52

3 CHAPTER A simple and sensitive assay for the quantitative analysis of rivastigmine and its metabolite AP in human EDTA plasma using coupled liquid chromatography and tandem mass spectrometry S.V. Frankfort, M. Ouwehand, M.J. van Maanen, H. Rosing, C.R. Tulner, J.H. Beijnen Abstract A sensitive and specific LC-MS/MS assay for the determination of rivastigmine and its major metabolite AP is presented. A 100 µl plasma aliquot was spiked with a structural analogue of rivastigmine as internal standard (PKF AE-1) and proteins were precipitated by adding 200 µl of methanol. After centrifugation 100 µl of the clear supernatant was mixed with 100 µl of methanol-water (30:70, v/v) and 25 µl-volumes were injected onto the HPLC system. Separation was acquired on a 150 x 2.0 mm ID Gemini C18 column using a gradient system with 10 mm ammonium hydroxide and methanol. Detection was performed by using a turboionspray interface and positive ion multiple reaction monitoring by tandem mass spectrometry. The assay quantifies rivastigmine from 0.25 to 50 ng/ml and its metabolite AP from 0.50 to 25 ng/ml, using 100 µl human plasma samples. Validation results demonstrate that rivastigmine and metabolite concentrations can be accurately and precisely quantified in human EDTA plasma. This assay is now used to support clinical pharmacologic studies with rivastigmine. Rapid Communications in Mass Spectrometry 2006;20:

4 Chapter Introduction Rivastigmine, ((S)- - ethyl-3- [(1- dimethyl- amino) ethyl] methyl - phenylcarbamate (Exelon )), an acetylcholinesterase inhibitor, has shown efficacy in the symptomatic treatment of mild to moderately severe Alzheimer s dementia (AD) 1,2. Rivastigmine interacts with acetylcholinesterase resulting in a carbamylated complex that slowly breaks down to form free enzyme. This temporary inhibition of acetylcholinesterase (AChE), leads to an increased availability of acetylcholine in cholinergic neurons of the brain 3. Cholinergic deficits lead to cognitive and behavioural disturbances in Alzheimer s disease 4 and increasing acetylcholine availability ameliorates these disturbances 5. Patients respond very differently to rivastigmine. Efficacy ranges from continuation of deterioration or maintaining baseline levels to a clear clinical effect 6,7. In addition, many patients discontinue therapy within 6 months due to experience of adverse events 8, most frequently nausea, vomiting and diarrhoea 9. Therefore, it would be ideal to support rivastigmine treatment in clinical practice with therapeutic drug monitoring (TDM) by measuring plasma concentrations and relating those to adverse events and efficacy. Rivastigmine is extensively metabolised by the target enzyme acetylcholinesterase to the decarbamylated metabolite AP ((S)-3-(1-dimethylamino-ethyl)-phenol). In an exvivo experiment in rat brain samples and plasma it was shown that increase in AP concentration correlated well with enzyme inhibition, so the concentration of AP reflects the extent of enzyme inhibition 10. Two methods are described in literature that measured rivastigmine and AP in plasma by liquid chromatography coupled to the tandem mass spectrometry (LC-MS/MS). Pommier et al. 11 developed a method which uses a volume of 0.5 ml heparinised plasma, stable isotopically labelled internal standards for both rivastigmine and AP , derivatisation of AP and extraction from plasma using liquid-liquid extraction (LLE). Enz et al. 10 also described a method in heparinised plasma. The internal standard in this method was not isotopically labelled and smaller volumes of plasma (0.1 ml) were used and rivastigmine and metabolite were extracted by a laborious LLE with ethylacetate from plasma. To support clinical studies in our hospital regarding TDM of rivastigmine, a sensitive LC- MS/MS assay was developed. Our aim was to develop a simple, rapid and sensitive method for measuring rivastigmine and AP in a matrix of EDTA plasma and to simplify the procedure for the extraction of the analytes from plasma. We achieved our goals successfully by developing a simple, sensitive and reproducible assay with protein precipitation (PP) as sample pre-treatment, using a structural analogue as internal standard instead of a non-commercial available isotopically labelled one. The assay has been successfully applied in clinical pharmacologic studies with rivastigmine. 54

5 Bioanalysis of rivastigmine and AP Experimental Materials Rivastigmine (C 14 H 22 2 O 2 ; Figure 1A), its metabolite AP (C 10 H 15 O, Figure 1B) and the internal standard PKF AE-1 (C 15 H 25 2 O 2, Figure 1C) were kindly supplied by ovartis Pharma AG (Basel, Switzerland). Methanol (LC gradient grade) was obtained from Bissolve Ltd. (Amsterdam, The etherlands). Ammonia 25% (analytical grade) was from Merck (Amsterdam, The etherlands). Distilled water was used throughout the analyses. Drug free human EDTA plasma was obtained from the Central Laboratory for Blood Transfusion (Sanquin, Amsterdam, The etherlands). Figure 1. Chemical structures of rivastigmine (A), AP (B) and internal standard PKF AE-1 (C). (A) O (B) (C) O OH O O Liquid Chromatograpy The HPLC system comprised an HP1100 (Agilent Technologies, Palo Alto, CA) binary pump, degasser and HP1100 (Agilent Technologies) autosampler. Separation was performed using a Gemini C18 column (150 x 2.0 mm ID, particle size 5 µm, Phenomenex, Torrance, CA, USA) with gradient elution. The mobile phase was set at a mixture of 10 mm ammonium hydroxide methanol (50:50, v/v) for 1 minute followed by a block gradient to 10 mm ammonium hydroxide in water methanol (5:95, v/v) in 0.1 minute, at which it was kept for 7 minutes. The flow-rate was set at 0.2 ml/min and 25 µl was injected. The autosampler temperature was set at 10 o C. 55

6 Chapter Mass spectrometry An API 2000 triple quadrupole MS equipped with an turbo-ionspray (Sciex, Thornhill, O, Canada) was used. Positive ion multiple reaction monitoring (MRM) was performed for selective and sensitive detection. Data were acquired and processed using the proprietary software application Analyst (version 1.2, Sciex, Canada). Mass transitions of m/z 251 to 206 and 166 to 121 were optimised for rivastigmine and AP226-90, respectively. Dwell times of 700 ms were used. Mass transition of m/z 263 to 218 was optimised for the internal standard PKF AE-1 with a dwell time of 700 ms. ebulizer and turbo gas (both compressed air) operated at 40 psi and 30 psi, respectively. The curtain gas was set at 35 psi and the collision gas ( 2 ) at 3 psi. The ionspray voltage was set at 5500 V, with a source temperature of 350 o C. Preparation of stock and working solutions Two sets of stock solutions of both rivastigmine and AP were prepared from independent weightings in methanol-water (30:70, v/v) at a concentration of 0.1 mg/ml. One stock solution was used to prepare calibration standards, the other to prepare quality control samples. Stock solutions were further diluted in methanol-water (30:70, v/v) to working solutions in a range of 10 to 10,000 ng/ml for preparing calibration standards for rivastigmine and in a range of 20 to 10,000 ng/ml for AP Additionally, stock solutions were further diluted in methanol-water (30:70, v/v) to working solutions in a range of 100 to 10,000 ng/ml for preparing quality control samples for rivastigmine and AP A stock solution of PKF AE-1 internal standard was prepared in water at a concentration of 0.1 mg/ml. The stock solution was further diluted with water to obtain a working solution of 100 ng/ml. Then this solution was further diluted in methanol to a final concentration of 5 ng/ml. All solutions were stored at 2-8 o C. Preparation of calibration standards and quality control samples in human plasma Control human EDTA plasma was centrifuged for approximately 5 minutes at 1,000 g. Calibration standards were prepared freshly in EDTA plasma in a range from 0.25 to 50 ng/ml for rivastigmine and from 0.50 to 25 ng/ml for AP , and vortex mixed for approximately 30 seconds before processing. Standards were processed and analysed in duplicate. Validation samples for rivastigmine and AP were spiked separately to control human EDTA plasma and stored at -20 o C. Concentrations of 0.25, 0.75, 15 and 40 ng/ml for rivastigmine and 0.5, 1.5, 10 and 20 ng/ml for AP were prepared. 56

7 Bioanalysis of rivastigmine and AP Sample preparation Plasma proteins were precipitated by adding 200 µl of methanol, which contained the internal standard in a concentration of 5 ng/ml, to 100 µl of sample. The samples were vortexed for 10 seconds and mixed for 10 min at 1250 rpm. ext, samples were centrifuged for 10 min at 23,100 g and 100 µl of the clear supernatant was mixed with 100 µl methanol-water (30:70, v/v) and transferred to a glass autosampler vial with insert. A volume of 25 µl was injected onto the HPLC column. Validation procedures Validation of the assay for quantitation of rivastigmine and AP in EDTA plasma included linearity, accuracy, precision, specificity, selectivity, ion suppression, recovery and stability 12. We prepared and analysed calibration standards in duplicate in three analytical runs. In order to establish the best weighting factor back-calculated calibration concentration was determined. The model with the lowest total bias and most constant bias across the range was considered the best fit. The linearity was evaluated by means of back-calculated concentrations of the calibration standards. The deviations from the nominal concentrations should be within ± 20% for the Lower Limit Of Quantitation (LLOQ) and within ± 15% for other concentrations with coefficient of variation (C.V.) values less than 20% and 15% for both the LLOQ and the other concentrations, respectively 12. Five replicates of the independently prepared QC samples in plasma were analysed together with calibration standards in three analytical runs. Accuracies were determined as the percentage difference of the measured concentration from the nominal concentration and the coefficient of variation was used to report the precision. The intra and inter-assay accuracies (% bias) should be within ±20% at the LLOQ level and within ±15% at the other concentrations 12. The intra and inter-assay precisions should be ±20% at the LLOQ level and ±15% at the other concentrations 12. To investigate whether endogenous matrix constituents interfered with the assay, six individual batches of control drug-free plasma samples containing neither analyte nor internal standard (double blank), samples containing only internal standard (blank), and LLOQ samples were prepared. Samples were processed according to the described procedures and analysed. Peak areas of compounds co-eluting with the analyte or internal standard should not exceed 20% of the analyte peak area at the LLOQ or 5% of the internal standard area. Deviations from the nominal concentrations should be within ± 20% 12. For the determination of ion suppression, control drug-free plasma was processed and dry extracts were dissolved with working solutions, containing the analytes and internal standard in methanol-water (1:1, v/v), that represented 100% recovery. Ion-suppression was determined by comparing the analytical response of these samples to that of the working 57

8 Chapter solutions, the loss of signal represents the ion-suppression. Recovery was determined by comparing the analytical response of processed QC samples with the analytical response of blank samples reconstituted with working solutions as described above. These experiments were performed in triplicate at three concentration levels. Overall recovery corresponded to the net response after subtraction of the ion-suppression and signal loss due to the extraction. Ion suppression and recovery experiments for the internal standard were performed in a similar way. The stability of rivastigmine and metabolite in spiked human EDTA plasma samples after 3 freeze-thaw cycles from nominally 20 C to ambient temperature was assessed in triplicate at 0.75 and 40 ng/ml for rivastigmine and 1.5 and 20 ng/ml for AP The mean rivastigmine and metabolite concentrations after 3 freeze-thaw cycles were compared against freshly prepared extracts. The stability of rivastigmine and its metabolite in spiked human EDTA plasma samples maintained at ambient temperature for 2 and 24 hours was evaluated at and 15 ng/ml for rivastigmine and 1.5 and 10 ng/ml for the metabolite. Stability was evaluated by comparing the rivastigmine and AP concentrations against freshly prepared extracts. Additionally, processed sample stability was assessed at 0.75 and 40 ng/ml for rivastigmine at 10 C in the autosampler after 3 days of storage and after 8 days of storage at 2-8 C by comparing the mean rivastigmine concentration against freshly prepared extracts. For the metabolite, processed sample stability was assessed at 1.5 and 20 ng/ml under the same conditions as described for rivastigmine. Finally, re-injection reproducibility of rivastigmine and its metabolite in human plasma was determined in duplicate at three concentration levels (0.75, 15 and 40 for rivastigmine; 1.5, 10 and 20 ng/ml for AP ) after 24 hours at nominally 10 C by comparing the mean concentrations against freshly prepared and analysed extracts. Rivastigmine and its metabolite are considered stable in biological matrix or extracts when % of the initial concentrations are found 12. Clinical Study The analytical method described in this article has been used to support a clinical study in patients with Alzheimer s disease or Lewy Body Dementia. In this study rivastigmine is twice-daily orally administrated. Daily doses of rivastigmine ranged between 3 and 12 mg. Blood samples were collected up to 7h after intake. After collection, blood samples were immediately centrifuged (1,000g for 10 min) and the plasma layer was stored at 20 o C until analysis. 58

9 Bioanalysis of rivastigmine and AP Results and discussion Method development LLE with ethyl acetate, based on the sample pre-treatment method described by Enz et al. 10, was tested. Plasma was mixed with 0.1 M sodium carbonate (ph 11) and extracted with ethyl acetate. Recoveries of approximately 60% were obtained. To increase the extraction recovery, diethyl ether and methyl-tert.-butyl ether were tested. With diethyl ether the recovery increased to 80%. However, a high variation in extraction recovery was observed for all tested extraction solvents, resulting in poor accuracies and precisions. Then protein precipitation with trichloro acetic acid, acetonitrile and methanol was tested. The use of trichloro acetic acid resulted in inclusion of the analytes in the protein precipitate. Protein precipitation with acetonitrile and methanol resulted in recoveries of approximately 60%. As the accuracy and precision after protein precipitation with methanol was better, it was chosen as sample pre-treatment for the extraction of rivastigmine and AP from human EDTA plasma. To obtain high sensitivity and selectivity MS/MS detection was chosen. The Q1 mass spectra of rivastigmine, AP and internal standard showed molecular ions [M+H] + at m/z 251, 166 and 263, respectively. Fragment ions, resulting from cleavage of the tertiary dimethyl amine moiety, were seen for rivastigmine, AP and internal standard at m/z 206, 121 and 218, respectively. Figure 2. Product ion spectrum of rivastigmine (precursor ion m/z 251). O 6.0E+07 O 206 Intensity (cps) 4.0E E E m/z The molecular ion of rivastigmine at m/z 251, of AP at m/z 166 and of internal standard at m/z 263 were used as precursor ions to generate the product ion spectra presented in figures 2, 3 and 4, for rivastigmine, AP and IS, respectively. The 59

10 Chapter main fragment ions of rivastigmine (m/z 206), AP (m/z 121) and internal standard (m/z 218) were most abundant for the analytes and were used for MRM. The proposed fragmentation patterns for rivastigmine, AP and internal standard are presented in figures 2, 3 and 4, respectively. Figure 3. Product ion spectrum of AP (precursor ion m/z 166). 2.0E OH Intensity (cps) 1.5E E E E m/z 121 Figure 4. Product ion spectrum of PKF AE-1 (precursor ion m/z 263). 1.0E+07 O O 218 Intensity (cps) 5.0E E m/z Two assays have described the quantitative analysis of rivastigmine and AP in biological fluids using LC-MS techniques 10,11. In these assays the analytes are chromatographically separated from matrix components using isocratic conditions and with mobile phases methanol-0.02 M ammonium acetate (55:45, v/v) 11 and acetronitrile-water (80:20, v/v) containing 0.1% formic acid. 10 In our laboratory, however, the best results were 60

11 Bioanalysis of rivastigmine and AP obtained with gradient elution with a mobile phase of 10 mm ammonium hydroxide in water methanol (50:50, v/v to 5:95, v/v). A basic mobile phase can be well suited for bioanalysis of basic compounds in combination with positive ionisation as shown earlier in our department for paclitaxel, an anticancer drug, and its metabolites 13,14. Higher intensities were shown for the main fragment ions of both rivastigmine and AP under basic conditions compared to acidic conditions (table 1). Due to the use of protein precipitation as sample pre-treatment it was necessary to use gradient elution in order to elute contaminants from the column separately from the analytes to prevent ion-suppression from endogenous compounds. Using this system, the retention time of rivastigmine was 6.2 min, of AP min and for the internal standard 6.0 min. Representative HPLC-MS/MS chromatograms of an LLOQ sample for rivastigmine, AP and the internal standard from control human plasma are depicted in figure 5. Table 1. Peak intensities for rivastigmine and AP and their product ions in acidic and basic mobile phase conditions. Compound (mass) Intensity (cps) Acidic (Formic Acid; 1µg/mL * ) Basic (Ammonium Hydroxide; 0.1 µg/ml * ) Rivastigmine (251) Product ion (206) AP (166) Product ion (121) * concentration of analytes (rivastigmine or AP ) Validation of the assay The assay was linear over a concentration range from 0.25 to 50.0 ng/ml for rivastigmine and ng/ml for the metabolite in human plasma. The linear regression of peak area ratio versus the concentration 1/x 2 (the reciprocal of the squared concentration) was weighted to obtain the lowest total bias and the most constant bias across the range. Correlation coefficients of the calibration curves for rivastigmine and metabolites respectively were always better than At all concentration levels deviation of measured concentrations from nominal concentration were between -7.0 and 2.8 % for rivastigmine and between -3.5 and 5.0% for AP CV values less than 8.8 and 10.8 % were obtained for rivastigmine and AP , respectively. Rivastigmine and AP proved to be stable in the supernatant after protein precipitation for 8 days when stored at 2-8 C. Processed samples were stable for 72h when stored at 10 C. Finally, re-injection reproducibility was established, the analytical run can be re-injected after at least 24 hours of storage in the autosampler (10 C). 61

12 Chapter Figure 5. Representative HPLC-MS/MS chromatograms of an LLOQ sample for rivastigmine (A, 0.25 ng/ml), AP (B, 0.5 ng/ml) and the internal standard from control plasma (C, 10.0 ng/ml). (A) Intensity (cps) (B) Intensity (cps) (C) Intensity (cps) time (min) time (min) time (min) Assay performance data for rivastigmine and its metabolite are summarised in tables 2A and 2B, respectively. The intra-assay accuracies (% bias) for rivastigmine were within ± 14.3% for the LLOQ and within ± 9.1% for other concentrations and found to be acceptable 12. The intra-assay accuracies (% bias) for AP were within ± 8.1% for the LLOQ and ±14.0 % for all concentrations. The intra-assay precisions for rivastigmine were less than 15.4 % at the LLOQ level and less than 10.4% for all other concentrations. The intra-assay precisions for its metabolite AP were less than 14.0 % at the 62

13 Bioanalysis of rivastigmine and AP LLOQ level and less than 14.4% for all concentrations. Thus, based upon acceptable accuracy and precision, the validated range for rivastigmine based on 100 µl of human plasma is from 0.25 to 50.0 ng/ml for rivastigmine and for the metabolite AP from 0.25 to 25.0 ng/ml. MRM chromatograms of six batches of control drug-free plasma contained no co-eluting peaks >20% of the rivastigmine and its metabolite area at the LLOQ level, and no coeluting peaks >5% of the area of internal standard. Deviations from the nominal concentrations at the LLOQ level were within ±19.6 % for rivastigmine. Deviations from the nominal concentrations at the LLOQ level for AP were within ± 15.3 %. The mean ion-suppression for rivastigmine and its metabolite were 9.4 and 19.9%, respectively. The mean ion-suppression for the internal standard was 17.3%. The mean recovery after protein precipitation was 70.9, 85.7, and 68.2% for rivastigmine, AP and the internal standard, respectively. Total recoveries obtained for rivastigmine, AP and internal standard were 63.8, 68.4, and 56.4 %, respectively. Stability data of rivastigmine and AP are presented in table 3A and 3B, respectively. Rivastigmine in not stable in EDTA plasma when kept at ambient temperatures for more than 2 hours. Less than 80% rivastigmine was recovered at low concentrations after 2 hours at ambient temperatures, whereas an increase of more than 20% was observed for AP Rivastigmine and AP are stable during 1 freeze/thaw cycle, however a decrease in rivastigmine and increase in AP was seen after 2 and 3 freeze/thaw cycles (data not shown). AP is formed by enzymecatalysed hydrolysis of rivastigmine. These cholinesterases are present in plasma and explain the degradation of rivastigmine in EDTA plasma when kept at ambient temperatures. Pommier et al. 11, added a competitive cholinesterase inhibitor (physostigmine hemisulphate) to heparin plasma to prevent degradation of rivastigmine. The ability to stabilise EDTA plasma with a competitive cholinesterase inhibitor should be further investigated. To prevent the ex-vivo hydrolysis of rivastigmine, samples obtained in the clinical study were immediately centrifuged and stored at -20 C within 30 minutes after collection and during analysis samples were processed directly after thawing. 63

14 Chapter Table 2A. Assay performance data for rivastigmine Run ominal concentration (ng/ml) Mean calculated concentration (ng/ml) Accuracy (% deviation) Precision (% CV) Average * 14.7 # Average * 7.9 # Average * 7.9 # Average * 9.6 # 15 * =Inter-assay accuracy(%), # =Inter-assay precision (%) umber of replicates Table 2B. Assay performance data for AP Run ominal concentration (ng/ml) Mean calculated concentration (ng/ml) Accuracy (% deviation) Precision (% CV) Average * 11.4 # Average * 13.9 # Average * 10.4 # Average * 14.4 # 15 * =Inter-assay accuracy(%), # =Inter-assay precision (%) umber of replicates 64

15 Bioanalysis of rivastigmine and AP Table 3A. Stability data for rivastigmine Conditions Matrix Initial conc (ng/ml) Found conc (ng/ml) Ambient, 2 h Plasma Ambient 24 h Plasma Freeze (-20 C)- thaw cycle Plasma C, 8 days Supernatant after PP Reinjection reproducibility Processed C, 24 h sample Autosampler, 10 C, 72 h Processed sample PP=protein precipitation Dev (%) CV (%) umber of replicates Table 3B. Stability data for AP Conditions Matrix Initial conc (ng/ml) Found conc (ng/ml) Ambient, 2 h Plasma Ambient 24 h Plasma Freeze (-20 C)- thaw cycle Plasma C, 8 days Supernatant after PP Reinjection reproducibility Processed C, 24 h sample Autosampler, 10 C 72 h Processed sample PP=protein precipitation Dev (%) CV (%) umber of replicates 65

16 Chapter Figure 6. Concentration vs. time profiles of rivastigmine and AP in a patient treated orally with twice daily 3 mg rivastigmine (A) and in a patient treated orally with twice daily 6 mg rivastigmine (B). (A) Concentration (ng/ml) Rivastigmine AP Time (h) (B) Concentration (ng/ml) Rivastigmine AP Time (h) 66

17 Bioanalysis of rivastigmine and AP Clinical study In figure 6, the concentration vs. time plots are presented for rivastigmine and AP from a patient treated orally with twice daily 3 mg rivastigmine and of a patient treated orally with twice daily 6 mg rivastigmine. A maximum rivastigmine concentration of 18.4 ng/ml was reached 0.5 hours after administration of the drug in the patient treated with twice daily 3 mg; the highest plasma level for AP was 6.2 ng/ml. The maximum concentration of rivastigmine (36.3 ng/ml) was reached 2 hours after administration of the drug in the patient treated with twice daily 6 mg; the highest plasma level for AP was 8.0 ng/ml. It is clear that rivastigmine levels in the patient treated with twice daily 6 mg are approximately twice the level compared to that in the patient treated with twice daily 3 mg. This, however, is not shown for the metabolite AP that reached maximum levels between 6 and 8 ng/ml for those 2 patients. Conclusions For the quantitation of rivastigmine and AP in human plasma, a simple, accurate, reproducible and selective LC-MS/MS assay has been developed. The assay quantifies a range for rivastigmine from 0.25 ng/ml to 50 ng/ml and for AP from 0.50 ng/ml to 25 ng/ml using 100 µl human plasma aliquots. Validation results demonstrate that the rivastigmine and metabolite concentrations can be accurately and precisely quantified in human plasma. This assay is now used to support clinical pharmacologic studies with rivastigmine. References 1. Corey-Bloom J, Anand R, Veach J. A randomized trial evaluating the efficacy and safety of EA 713 (rivastigmine tartrate), a new acetylcholinesterase inhibitor, in patients with mild to moderately severe Alzheimer s disease. International Journal of Geriatric Psychopharmacology 1998;1: Rösler M, Anand R, Cican-Sain A, et al. Efficacy and safety of rivastigmine in patients with Alzheimer s disease: international randomised controlled trial. BMJ 1999; 318: Jann MW, Shirley KL, Small GW. Clinical pharmacokinetics and pharmacodynamics of cholinesterase inhibitors. Clin Pharmacokinet 2002;41: Francis PT, Palmer AM, Snape M, Wilcock G. The cholinergic hypothesis of Alzheimer's disease: a review of progress. J eurol eurosurg Psychiatry 1999;66: Davis KL, Mohs RC, Marin D, et al. Cholinergic markers in elderly patients with early signs of Alzheimer s disease. JAMA 1999;281: Rockwood K, MacKnight C. Assessing the clinical importance of statistically significant improvement in anti-dementia drug trials. euroepidemiology 2001;20: Frankfort SV, Appels BA, de Boer A, et al. Treatment effects of rivastigmine on cognition, performance of daily living activities and behaviour in Alzheimer s disease in an outpatient geriatric setting. Int J Clin Pract 2006;60: Frankfort SV, Appels BA, de Boer A, et al. Discontinuation of rivastigmine in routine clinical practice. Int J Geriatr Psychiatry 2005;20:

18 Chapter Gauthier S. Cholinergic adverse effects of cholinesterase inhibitors in Alzheimer s disease. Drugs Aging 2001;18: Enz A, Chappuis A, Dattler A. A simple, rapid and sensitive method for simultaneous determination of rivastigmine and its major metabolite AP in rat brain and plasma by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry. Biomed Chromatogr 2004;18: Pommier F, Frigola R. Quantitative determination of rivastigmine and its major metabolite in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry J Chromatogr B Analyt Technol Biomed Life Sci 2003;784: Rosing H, Man WY, Doyle E, Bult A, Beijnen JH. Bioanalytical liquid chromatographic method validation. A review of current practices and procedures. J Liq Chrom Rel Technol 2000; 23: Vainchtein LD, Thijssen B, Stokvis E, et al. A simple and sensitive assay for the quantitative analysis of paclitaxel and metabolites in human plasma using liquid chromatography /tandem mass spectrometry. Biomed Chromatogr 2006;20: Stokvis E, Ouwehand M, an LGAH, et al. A simple and sensitive assay for the quantitative analysis of paclitaxel in human and mouse plasma and brain tumor tissue using coupled liquid chromatography and mass spectrometry. J Mass Spectr 2004;39: