Effects of nebulizing and drying gas flow on capillary electrophoresis/mass spectrometry

This study was focused on examining the influence of gas flow parameters on capillary electrophoresis/mass spectrometry (CE /MS) performance using sheath-liquid CE /MS interfaces. The effects of nebulizing and drying gas velocity and drying gas temperature on CE separation and MS detection sensitivity were systematically determined. Nebulizing gas velocity was observed to be a critical parameter in the optimization of CE /MS method, since it affected both MS detection sensitivity, and also CE separation efficiency for one interface design tested. Better detection sensitivity was obtained when the nebulizing gas velocity was increased. However, high velocity of the nebulizing gas flow can cause a hydrodynamic bulk flow inside the CE capillary, thus clearly increasing the apparent mobility and decreasing the resolution obtained for the compounds studied. Increasing the drying gas velocity or temperature did not affect the apparent mobility or the separation efficiency and the temperature could be increased to achieve the optimal detection sensitivity in the CE /MS analysis. For comparison, the effects of nebulizing gas flow were studied using a different design of the coaxial sheath-liquid CE /MS interface, and in this case better detection sensitivity but no effect on CE separation efficiency was observed with increased nebulizing gas velocity. These different effects of nebulizing gas flow on the CE bulk flow were concluded to result from pressure differences at the tip of the CE capillaries for the different CE /MS interface arrangements. It is therefore recommended that the cross-sectional dimensions of the fused-silica and steel capillaries, and the gas streamlines, should be optimized when CE /MS interfaces are built. Moreover, the effect of gas flow on CE separation should be studied when optimizing the CE /MS operation parameters.     

Analyses of alkaloids in different products by NACE-MS

A simple method for the separation and characterization of five nicotine-related alkaloids by NACE employing UV and MS detections is described here for the first time. Several factors, including NACE parameters (compositions of running solution) and MS parameters (such as nature and flow rate of sheath liquid, pressure of nebulization gas, and flow rate of dry gas), were optimized in order to obtain both an adequate CE separation and high MS signals for the alkaloid compounds used in this study. A reliable CE separation of five alkaloids was achieved in 50 mM ammonium formate that was dissolved in an ACN/methanol mixture (50:50, v/v) of pH* 4.0 (apparent pH 4.0). The optimal electrospray MS measurement was carried out in the positive ionization mode using a coaxial sheath liquid composed of isopropyl alcohol and water in the ratio of 80:20 v/v at a flow rate of 180 microL/h. In addition, the proposed NACE method was also applied in the analyses of alkaloids in several products including chewing gums, beverages, and tobaccos. This NACE-MS method was found to provide a better detection ability and separation resolution for the analysis of nicotine alkaloids when compared to other aqueous CE-MS reports.     

Metabolite profiling of human urine by CE-ESI-MS using separation electrolytes at low pH

We investigated the potential of CE coupled to electrospray MS (CE-ESI-MS) in metabolite profiling of human urine without any sample prefractionation step. A heterogeneous mixture of biologically relevant compounds covering a broad range of physicochemical properties was used to optimize separation conditions in fused-silica capillaries. A running electrolyte containing 50 mM of acetic acid and 50 mM of formic acid at pH 2.5 was used for the CE separations. A sheath-flow electrospray interface was employed for CE-ESI-MS analysis. Sheath liquids containing 80:20 v/v methanol/water with 0.1% v/v of acetic acid or 60:40 v/v isopropanol/water with 0.5% v/v of ammonia were selected for optimum detection in positive and negative ESI modes, respectively. Reproducibility and sensitivity were studied, and strategies for identification of the separated urinary compounds are suggested. We report major advantages and disadvantages of CE-ESI-MS for metabolite profiling of human body fluids. This work may be regarded as a first step in the use of CE-ESI-MS for reliable differential analysis of body fluids from healthy and diseased individuals.     

CE-ESI-MS separation of divalent organic and inorganic anions using a tricationic complexing reagent

A tricationic ion-pairing reagent, 1,3,5-1-butyl-3-methyl-1H-imidazol-3-ium-2,4,6-trimethylbenzene, was used to form complexes with doubly charged anions for their subsequent analysis by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in the positive-ion mode. This methodology offers the advantages of greater versatility and sensitivity relative to direct detection of the anions in negative-ion mode, and it can be realized by a number of possible complexation strategies, including pre-column, on-column, and post-column modes. Three model anions, sulfate [SO?2?], thiosulfate [TSFA, S?O?2?], and benzenedisulfonate [BZDS, C?H?(SO?)?2?], were amenable to complexation with the tricationic reagent, yielding singly charged cations with greater mass-to-charge (m/z) ratios than the native analytes. By utilizing optimized parameters obtained through previous work with dicationic reagents and singly charged anions, including the CE separation buffer composition and pH, the concentration of the dicationic reagent, the mode of complexation, the nebulizing gas pressure, and the sheath liquid composition, it was possible to develop a robust CE-ESI-MS method appropriate for the analysis of divalent anions in a mixture.     

Optimization of CE-ESI-MS Parameters for the Analysis of Ecstasy and Derivatives in Urine

Capillary electrophoresis (CE) was interfaced with a quadrupole mass spectrometer, using pneumatically-assisted electrospray ionization (ESI) in order to analyze Ecstasy and derivatives in urine. The influence of the sheath liquid composition, including the type and the percentage of the organic solvent, as well as the nature of the conductive acid modifier, were examined in order to find optimal coupling conditions. A fractional factorial design was also used to optimize the electrospray experimental parameters, such as the nebulizing gas pressure, the electrospray voltage, the drying gas flow rate, the drying gas temperature, the skimmer voltage, and the sheath liquid flow rate. The separation conditions were optimized in terms of temperature, electrolyte concentration, percentage of organic modifier, as well as capillary type. Finally, the optimal CE-ESI-MS conditions were applied to the analysis of Ecstasy and other related amphetamines in urine samples, following a liquid-liquid extraction procedure.     

Analysis of benzalkonium chloride by capillary electrophoresis-tandem mass spectrometry

Conditions for the separation and determination of benzalkonium chloride (BAC) homologues by CE with UV-detection and CE coupled to MS (IT) using electrospray as ionization source were established. The separation was performed using fused-silica capillaries of 50 microm id and 100 mM acetic acid-ammonium acetate buffer solution at pH 4.5 with 80% of ACN as carrier electrolyte. CE-MS coupling parameters were optimized and methanol-10 mM acetic acid (90:10 v/v) was selected as sheath liquid. Detection limits, based on an S/N of 3:1, were calculated, and values between 0.8 and 1.3 mg/L with CE-ESI/MS and around 0.5 mg/L with CE-ESI-MS/MS, using hydrodynamic injection (15 s, 3.5 kPa), were obtained. Good run-to-run and day-to-day precisions on concentration were achieved with RSDs lower than 8%. Quantitative analysis was carried out by the internal standard method and the calibration curves showed good linearities (r(2) > 0.98). The CE-ESI-MS/MS method was successfully applied to the analysis of BAC in different ophthalmic solutions, allowing the direct determination, identification and confirmation of the BAC homologues presented in these samples.     

Positional and geometrical anionic isomer separations by capillary electrophoresis-electrospray ionization-mass spectrometry

Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) was applied to the analysis of polar positional and geometrical anionic isomers. Since the investigated positional and geometrical anionic isomers have different pK(a) values, they could be separated by CE-ESI-MS under simple analytical conditions using a bare fused-silica capillary and volatile ammonium acetate buffer after optimizing buffer pH. Ortho-, meta-, para-hydroxybenzoate positional isomers were completely separated on a fused-silica capillary with 20 mM ammonium acetate buffer at pH 10.0, and cis-, trans-cyclohexane dicarboxylate geometrical isomers could be also separated with 20 mM ammonium acetate buffer at pH 4.0. Several analytical parameters affecting ESI-MS sensitivity were also investigated. It was found that both running buffer pH and sheath liquid pH had significant effects on the selectivity and the sensitivity on CE-ESI-MS analysis while sheath flow rate and other parameters had little influence. Under optimized conditions, linearity, detection limit, and repeatability of the analysis of hydroxybenzoate isomers were examined, and good results were obtained. It was found that the method presented in this paper is a simple, robust, and cost-effective method for simultaneous analysis of positional and geometrical anionic isomers as well as of other small anionic compounds.     

Development of a CE-ESI-microTOF-MS method for a rapid identification of phenolic compounds in buckwheat

A sensitive CE-ESI-MS analytical method for the identification of buckwheat antioxidants has been developed. CE and ESI-TOF parameters (e.g. buffer composition and pH, sheath liquid composition, sheath liquid and gas flow rates, electrospray voltage) were optimized to obtain an optimal analytical separation and identification. The results confirmed the presence of phenolic acids, procyanidins and galloylated propelargonidins. The identification of swertiamacroside and 2-hydroxy-3-O-β-D-glucopyranosil-benzoic acid, found for the first time in our previous work, has been confirmed. Furthermore, 5,7,4'-trimethoxyflavan and dihydroxy-trimethoxyisoflavan have also been tentatively identified for the first time in buckwheat.     

CE-ESI-MS of biological anions in plastic capillaries at high pH

In this study, the potential of poly(methylmethacrylate) (PMMA, Plexiglas) and polyether ether ketone (PEEK) tubing for CE-ESI-MS separations of anions at high pH values was examined. A set of model compounds of biological interest was used to investigate the main operational parameters for CE-ESI-MS, such as the sheath-flow interface design, the polarity of the ionization voltage, the use of ammonia-based separation electrolytes, and the sheath liquid composition. Optimum separations and detection sensitivities in negative ESI mode were obtained using a running electrolyte of 75 mM of ammonia at pH 11 and a sheath liquid of 60:40 v/v or 75:25 v/v isopropanol/water with 0.5% v/v of ammonia. At these experimental conditions, PMMA and PEEK capillaries show good hydrolytic stabilities and lower EOF values than fused-silica columns. Better separation resolutions were obtained with PMMA capillary, but this plastic rapidly swelled and bled because of its limited chemical resistance to the sheath liquid. PMMA columns equipped with a fused-silica tip were used for a safer exposure to the sheath liquid, but the inner surface of the fused-silica tips had limited stability at pH 11. On the other hand, good separations and reproducibility on migration times and peak areas were obtained using PEEK capillaries without capillary column deterioration.     

A new method for screening and determination of diuretics by on-line CE-ESI-MS

A rapid, high-resolution and effective new method for analyzing 12 diuretics by CE-ESI-MS was established in this paper. Ten diuretics (except two neutral compounds) could be fast separated by CE with a DAD at 214 nm with a 20 kV voltage within 6 min, using a 50 microm id and 48.5 cm effective length uncoated fused-silica capillary in a 40 mM ammonium formate buffer (pH 9.40). CE was coupled to the mass spectrometer applying an orthogonal electrospray interface with a triple-tube sheath liquid arrangement. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 30 mM acetic acid with a flow rate of 4 microL/min. Mass spectrum was employed in the positive mode and both full scan mode and SIM scan mode were utilized. All 12 diuretics could be detected and confirmed by MS in a single analysis. Under optimized conditions, LODs for the 12 diuretics were in the range of 0.13-2.7 micromol/L at an S/N of 3, and the correlation coefficients R(2 )were between 0.9921 and 0.9978. The RDSs (n = 5) of the method was 0.24-0.94 % for migration times and 1.6-8.8 % for peak areas. The recoveries of spiked samples of 12 diuretics were between 72.4% and 118%. The real urine samples were injected directly for analysis, with only simple filtration through a 0.22 microm membrane filter in order to remove solid particles, which may cause capillary blockage. Based on the migration times and characteristic ions, the diuretics in urine samples were detected successfully. This CE-ESI-MS method for analyzing diuretics will hopefully be applied to doping control.     

Capillary electrophoresis-electrospray ionization-mass spectrometry method to determine the phenolic fraction of extra-virgin olive oil

We describe the first analytical method involving SPE and CZE coupled to ESI-IT MS (CZE-ESI-MS) used to identify and characterize phenolic compounds in olive oil samples. The SPE, CZE and ESI-MS parameters were optimized in order to maximize the number of phenolic compounds detected and the sensitivity of their determination. To this end we have devised a detailed method to find the best conditions for CE separation and the detection by MS of the phenolic compounds present in olive oil using a methanol-water extract of Picual extra-virgin olive oil (VOO). Electrophoretic separation was carried out using an aqueous CE buffer system consisting of 60 mM NH(4)OAc at pH 9.5 with 5% of 2-propanol, a sheath liquid containing 2-propanol/water 60:40 v/v and 0.1% v/v triethylamine. This method offers to the analyst the chance to study important phenolic compounds such as phenolic alcohols (tyrosol (TY), hydroxytyrosol (HYTY) and 2-(4-hydroxyphenyl)ethyl acetate), lignans ((+)-pinoresinol and (+)-1-acetoxypinoresinol), complex phenols (ligstroside aglycon (Lig Agl), oleuropein aglycon, their respective decarboxylated derivatives and several isomeric forms of these (dialdehydic form of oleuropein aglycon, dialdehydic form of ligstroside aglycon, dialdehydic form of decarboxymethyl elenolic acid linked to HYTY, dialdehydic form of decarboxymethyl elenolic acid linked to TY) and 10-hydroxy-oleuropein aglycon) and one other phenolic compound (elenolic acid) in extra-VOO by using a simple SPE before CE-ESI-MS analysis.     

Utility of nonaqueous capillary electrophoresis for the determination of lidocaine and its metabolites in human plasma: a comparison of ultraviolet and mass spectrometric detection

A nonaqueous capillary electrophoresis/electrospray mass spectrometry method for the separation of lidocaine (LID) and two of its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), has been developed. The separation medium was: 70 mM ammonium formate and 2.0 M formic acid in acetonitrile/methanol (60:40 v/v). With a sheath liquid of methanol/water (80:20 v/v) containing 2% formic acid and positive ion detection, reproducible determinations (8-11% relative standard deviation (RSD)) of lidocaine and its metabolites were performed in spiked human plasma. The limits of detection (LODs) were between 69.1 and 337 nM. The influences of sheath liquid composition, nebulizing gas pressure and drying gas temperature on the separation were examined.     

Determination of stimulants and narcotics as well as their in vitro metabolites by online CE-ESI-MS

A simple, rapid and sensitive CE-ESI-MS method for the simultaneous analysis of seven stimulants and narcotics (amphetamine, ephedrine, methadone, pethidine, tetracaine, codeine and heroin) was developed. The CE-ESI-MS experimental conditions were optimized as follows: 20?mmol/L ammonium acetate with pH 9.0 as running buffer, the separation voltage of 22?kV and the sheath liquid of isopropanol/water (1:1 v/v) containing 7.5?mmol/L acetic acid with 3.0?μL/min flow rate. Under the optimized conditions, the stimulants and narcotics were well separated within 4.6?min using a 70-cm length fused-silica capillary (50?μm id). The detection limits (S/N=3) of the CE-ESI-MS analysis were in the range of 0.40-1.0?ng/mL. Method repeatability of intra-day and inter-day was satisfactory. The recoveries obtained from the analysis of spiked urine samples were between 84.1 and 108%. The developed method was successfully applied for the simultaneous analysis of methadone, pethidine and codeine and their in vitro metabolites.     

Nonaqueous capillary electrophoresis-electrospray- mass spectrometry for the analysis of fluoxetine and its related compounds

The potential of nonaqueous capillary electrophoresis was investigated for the simultaneous separation of fluoxetine hydrochloride, its meta-isomer, and other related compounds. The resolution of these compounds was compared in aqueous and nonaqueous media. Baseline separation of the studied solutes required a buffer electrolyte solution composed of 25 mM ammonium acetate and 1 M acetic acid in acetonitrile, an applied voltage of 30 kV and a temperature of 20 degrees C. Selectivity was considerably affected by the nature of the solvent (water, methanol, and acetonitrile). Moreover, substituting acetate by formate in the background electrolyte resulted in migration time changes, which were attributed to an ion-pairing phenomenon. Finally, the method was successfully coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) and allowed significant selectivity and sensitivity enhancement. The effect of ESI-MS parameters, such as nebulizing gas pressure, sheath liquid composition and flow rate, on resolution and method sensitivity was also discussed.     

Simultaneous chiral separation and determination of ephedrine alkaloids by MEKC-ESI-MS using polymeric surfactant I: method development

In this work, simultaneous separation of eight stereoisomers of ephedrine and related compounds ((+/-)-ephedrine, (+/-)-pseudoephedrine, (+/-)-norephedrine and (+/-)-N-methylephedrine) was accomplished using a polymeric chiral surfactant, i.e. polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) by chiral (C)MEKC-ESI-MS. The conditions of CMEKC were first investigated. The baseline separation of all eight stereoisomers of ephedrine and related compounds was achieved under optimum CMEKC conditions (35 mM poly-L-SUCL, 15 mM NH(4)OAc, pH 6.0, 30% v/v ACN, 30 kV and 20 degrees C) in less than 30 min. Next, a central composite design for response surface modeling has been described to evaluate the electrospray chamber parameters and the sheath liquid conditions. Optimum mass abundance of stereoisomers of ephedrine and related compounds was observed using the spray chamber parameters, namely 250 degrees C drying gas temperature and 8 L/min drying gas flow rate at a nebulizer pressure of 4 psi. Furthermore, the experimental design indicates that the optimum mass abundance of the stereoisomers of ephedrine and related compounds can be obtained using a sheath liquid containing 80:20 v/v methanol-water, 5 mM NH(4)OAc at pH 8.5 delivered at 5 microL/min. Finally, compared to MEKC-UV, the use of poly-L-SUCL in MEKC-MS provided significantly higher sensitivity for stereoisomers of ephedrine and related compounds.     

Optimization of capillary electrophoretic-electrospray ionization-mass spectrometric analysis of catecholamines

The capillary electrophoretic-mass spectrometric analysis (CE-MS) of catecholamines was optimized with coaxial sheath flow interface and electrospray ionization (ESI). The parameters studied included the sheath liquid composition and its flow rate, separation conditions in ammonium acetate buffer together with the ESI and cone voltages as mass spectrometric parameters. In addition, the effect of ESI voltage on injection as well as the siphoning effect were considered. The optimized conditions were a sheath liquid composition of methanol-water (80:20 v/v) with 0.5% acetic acid, with a flow rate of 6 microL/min. The capillary electrophoretic separation parameters were optimized with 50 mM ammonium acetate buffer, pH 4.0, to +25 kV separation voltage together with a pressure of 0.1 psi. The most intensive signals were obtained with an ESI voltage of +4.0 kV and a cone voltage of +20 V. The nonactive ESI voltage during injection as well as avoidance of the siphoning effect increased the sensitivity of the MS detection considerably. The use of ammonium hydroxide as the CE capillary conditioning solution instead of sodium hydroxide did not affect the CE-MS performance, but allowed the conditioning of the capillary between analyses to be performed in the MS without contaminating the ion source.     

Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS. The influence of several CE and MS operating parameters, such as the concentration of CE running buffer, applied external pressure, and the composition and flow rate of sheath liquid on CE-ESI-MS was studied. The results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate running buffer was 4.80 and 5.50, respectively, a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In the online CE-ESI-MS experiments, we not only achieved the online MS signal of uEPO, but also obtained baseline separation of three major rhEPO glycoforms successfully and reproducibly on the 6,6-ionene-coated capillaries. Furthermore, the standard mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. Additionally, the molecular weight of rhEPO and uEPO were verified and compared to the results by MALDI-TOF-MS. It can be concluded that, in contrast to other indirect methods, the online CE-ESI-MS technique with the combination of the advantages of both CE and MS shows great potential for the separation and detection of rhEPO doping directly in competitive sports.     

Coupling of capillary electrophoresis with electrospray ionization multiplexing ion mobility spectrometry

In this work, ion mobility spectrometry (IMS) function as a detector and another dimension of separation was coupled with CE to achieve two‐dimensional separation. To improve the performance of hyphenated CE‐IMS instrument, electrospray ionization correlation ion mobility spectrometry is evaluated and compared with traditional signal averaging data acquisition method using tetraalkylammonium bromide compounds. The effect of various parameters on the separation including sample introduction, sheath fluid of CE and drift gas, data acquisition method of IMS were investigated. The experimental result shows that the optimal conditions are as follows: hydrodynamic sample injection method, the electrophoresis voltage is 10 kilo volts, 5 mmol/L ammonium acetate buffer solution containing 80% acetonitrile as both the background electrolyte and the electrospray ionization sheath fluid, the ESI liquid flow rate is 4.5 μL/min, the drift voltage is 10.5 kilo volts, the drift gas temperature is 383 K and the drift gas flow rate is 300 mL/min. Under the above conditions, the mixture standards of seven tetraalkylammoniums can be completely separated within 10 min both by CE and IMS. The linear range was 5–250 μg/mL, with LOD of 0.152, 0.204, 0.277, 0.382, 0.466, 0.623 and 0.892 μg/mL, respectively. Compared with traditional capillary electrophoresis detection methods, the developed CE‐ESI‐IMS method not only provide two sets of qualitative parameters including electrophoresis migration time and ion drift time, ion mobility spectrometer can also provide an additional dimension of separation and could apply to the detection ultra‐violet transparent compounds or none fluorescent compounds.     

Pressurized liquid extraction of lipids for the determination of oxysterols in egg-containing food

Pressurized liquid extraction (PLE, ASE) was compared with the Folch procedure (a solid-liquid extraction with chloroform/methanol 2:1, v/v) for the lipid extraction of egg-containing food; the accuracy of PLE for the quantitative determination of oxysterols in whole egg powder was evaluated. Samples of spray-dried whole egg, an Italian vanilla cake (Pandoro) and egg noodles were used. Two different extraction solvents (chloroform/methanol 2:1, v/v, and hexane/isopropanol 3:2, v/v) were tested at different extraction temperatures and pressures (60 degrees C at 15 MPa, 100 degrees C at 15 MPa, 120 degrees C at 20 MPa). No significant differences in the lipid recovery of the egg powder sample using PLE were found. However, PLE of the vanilla cake and egg noodles with the chloroform/methanol mixture was not selective enough and led to the extraction of a non-lipid fraction, including nitrogen-containing compounds. In the same samples, the pressurized hexane/isopropanol mixture gave a better recovery result, comparable to that obtained using the Folch method. Cholesterol oxidation products of the Folch extract and the pressurized liquid extract of spray dried egg powder (obtained with hexane/isopropanol 3:2, v/v, at 60 degrees C and 15 MPa) were determined by gas chromatography. PLE performed under these conditions is suitable to replace the Folch extraction, because the differences between the two methods tested were not statistically significant. Moreover, PLE shows important advantages, since the analysis time was shortened by a factor of 10, the solvent costs were reduced by 80% and the use of chlorinated solvents was avoided.     

Determination of berberine and strychnine in medicinal plants and herbal preparations by pressurized liquid extraction with capillary zone electrophoresis.

A simple and rapid method for the determination of berberine and strychnine in medicinal plants and herbal preparations for regulatory purposes using a home-made pressurized liquid extraction (PLE) system with capillary zone electrophoresis (CZE) using ultraviolet detection at 254 nm was developed. The effects of pH, concentration of buffer, and organic modifiers in the electrophoretic separation were investigated. The buffer used for CZE contained 50 mM ammonium acetate, pH 3.1. The effect of temperature on the extraction efficiency of strychnine in medicinal plants by PLE was demonstrated. Comparable or higher extraction efficiency was achieved with PLE for strychnine in medicinal plants and berberine in herbal preparations compared to soxhlet extraction. The effect of matrix interference in medicinal plants and herbal preparations containing a number of medicinal plants samples using CZE was investigated by standard additional experiments. The reproducibility of the method using PLE with CZE was found to vary between 2.4 and 10.7% (n = 5/6) for different types of samples on different days.