Chemiluminescence selectivity enhancement in the on-chip Ru(bpy)32+ system: The potential role of buffer type and pH in the determination of hydrochlorothiazide in combined formulations and human plasma.

A simple and highly selective on-chip Ru(bpy)32+ -oxidant chemiluminescence (CL) approach for estimation of a diuretic drug, hydrochlorothiazide (HCZ), in biological fluids was realized in the presence of other fixed-dose combination drugs by manipulating simultaneously the method of active species (Ru(bpy)33+ ) production and type of carrier buffer with pH used for the CL reaction. Chemical oxidation processes involved in the Ru(bpy)32+ -Ce(IV) CL system have been successfully miniaturised in this study using a microfabricated device to generate Ru(bpy)33+ instantaneously. The proposed system was then screened using HCZ and other drugs in the presence of various buffers and pH to explore the difference in CL emission. Ammonium formate buffer (0.15 M) at pH 4.5 exhibited excellent selectivity towards HCZ when Ru(bpy)33+ was produced by chemical oxidation using Ce(IV). The newly developed conditions do not involve any kind of prior separation or isolation procedure to remove other combination therapy drugs in formulation and biological samples. The method under fully optimised conditions exhibited wide linearity over the concentration range 0.5-1000 ng ml-1 and low detection and quantification limits of 0.13 and 0.47 ng ml-1 respectively for HCZ. Acceptable levels of recoveries were obtained for HCZ from human plasma using the proposed method (98.9-100.8%) in the presence of other antihypertensive combination therapy drugs. This study postulates that such miniaturised devices may find applications especially for on-site analysis, such as doping control examinations.

Microfluidic photoinduced chemical oxidation for Ru(bpy)33+ chemiluminescence - A comprehensive experimental comparison with on-chip direct chemical oxidation.

For the first time, the analytical figures of merit in detection capabilities of the very less explored photoinduced chemical oxidation method for Ru(bpy)32+ CL has been investigated in detail using 32 structurally different analytes. It was carried out on-chip using peroxydisulphate and visible light and compared with well-known direct chemical oxidation approaches using Ce(IV). The analytes belong to various chemical classes such as tertiary amine, secondary amine, sulphonamide, betalactam, thiol and benzothiadiazine. Influence of detection environment on CL emission with respect to method of oxidation was evaluated by changing the buffers and pH. The photoinduced chemical oxidation exhibited more universal nature for Ru(bpy)32+ CL in detection towards selected analytes. No additional enhancers, reagents, or modification in instrumental configuration were required. Wide detectability and enhanced emission has been observed for analytes from all the chemical classes when photoinduced chemical oxidation was employed. Some of these analytes are reported for the first time under photoinduced chemical oxidation like compounds from sulphonamide, betalactam, thiol and benzothiadiazine class. On the other hand, many of the selected analytes including tertiary and secondary amines such as cetirizine, azithromycin fexofenadine and proline did not produced any analytically useful CL signal (S/N=3 or above for 1µgmL-1 analyte) under chemical oxidation. The most fascinating observations was in the detection limits; for example ofloxacin was 15 times more intense with a detection limit of 5.81×10-10M compared to most lowest ever reported 6×10-9M. Earlier, penicillamine was detected at 0.1µgmL-1 after derivatization using photoinduced chemical oxidation, but in this study, we improved it to 5.82ngmL-1 without any prior derivatization. The detection limits of many other analytes were also found to be improved by several orders of magnitude under photoinduced chemical oxidation.

Parallel microdevice for high throughput analysis of levofloxacin using tris (2,2'-bipyridyl) ruthenium (II) and peroxydisulfate chemiluminescence system.

A parallel microdevice has been developed for high throughput analysis using microfluidics. The detection method is based on a chemiluminescence (CL) system based on the oxidation of tris (2,2'-bipyridyl) Ru(ll) [Ru (bipy)3(2+)] by peroxydisulfate. The device consists of a photoreactor chip and two detection chips. The sample throughput can reach up to 720 runs/h with the total reagent consumption of only 2.4 mL. The parallel microdevice was evaluated using levofloxacin (LEVO) in pharmaceutical preparations. The various factors that affect the CL signal were optimized, and the LOD was found to be 30 and 27 microg/L for the two detectors, respectively (S/N = 3), while RSD was 1.1% (n = 15) for 1.0 mg/L LEVO. Two tablet samples that contain LEVO as an active ingredient were successfully analyzed using the proposed parallel microdevice.

A lab-on-a-chip device for analysis of amlodipine in biological fluids using peroxyoxalate chemiluminescence system.

A highly sensitive, rapid and economical method for the determination of amlodipine (AM) in biological fluids was developed using a peroxyoxalate chemiluminescence (CL) system in a lab-on-a-chip device. Peroxyoxalate-CL is an indirect type of CL that allows the detection of native fluorophores or compounds derivatized with fluorescent labels. Here, fluorescamine was reacted with AM, and the derivatization product was used in a bis-(2,4,6-trichlorophenyl)oxalate-CL system. Fluorescamine reacts selectively with aliphatic primary amine at neutral or basic pH. As most of the calcium channel blocker and many cardiovascular drugs do not contain primary amine, the developed method is highly selective. The parameters that influenced the CL signal intensity were studied carefully. These included the chip geometry, pH, concentration of reagents used and flow rates. Moreover, we confirmed our previous observation about the effects of imidazole, which is commonly used in the bis-(2,4,6-trichlorophenyl)oxalate-CL system as a catalyst, and found that the signal was significantly improved when imidazole was absent. Under optimized conditions, a calibration curve was obtained with a linear range (10-100 µg/L). The limit of detection was 3 µg/L, while the limit of quantification was 10 µg/L. Finally the method was applied for the determination of AM in biological fluids successfully.

Determination of amlodipine using terbium-sensitized luminescence in the presence of europium(III) as a co-luminescence reagent.

A sensitive time-resolved luminescence method for the determination of amlodipine (AM) in methanol and in aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb(3+) ) by formation of a ternary complex with AM in the presence of tri-n-octylphosphine oxide (TOPO) as co-ligand, dodecylbenzenesulfate as surfactant and europium ion as a co-luminescence reagent. The signal for Tb-AM-TOPO is monitored at λex = 242 nm and λem = 550 nm. Optimum conditions for the formation of the complex in aqueous system were 0.015 m Tris (hydroxylmethyl) amino methane buffer, pH 9.0, TOPO (1.0 × 10(-4) m), Eu(3+) (2.0 × 10(-7) m), dodecylbenzenesulfate (0.14%) and 6.0 × 10(-5) m of Tb(3+) , which allows the determination of 10-50 ppb of AM with a limit of detection of 1.2 ppb. The relative standard deviations of the method range between 0.1 and 0.2% indicated excellent reproducibility of the method. The proposed method was successfully applied for the assay of AM in pharmaceutical formulations and in plasma samples. Average recoveries of 98.5 ± 0.2% and 95.2 ± 0.2% were obtained for AM in tablet and plasma samples respectively.

High-throughput method for the analysis of venlafaxine in pharmaceutical formulations and biological fluids, using a tris(2,2'-bipyridyl) ruthenium(II)-peroxydisulphate chemiluminescence system in a two-chip device.

A simple, rapid and sensitive chemiluminescent (CL) method for the assay of venlafaxine (VEN) in pharmaceutical formulations and serum samples by a two-chip device is proposed. The method is based on the reaction of this drug with a tris(2,2'-bipyridyl) ruthenium(II)-peroxydisulphate CL system. The optimum chemical conditions for CL emission were investigated. The calibration graph was linear for the concentration range 0.02-8.0 µg/mL. The detection and quantification limits were found to be 0.006 and 0.018 µg/mL, respectively, while the relative standard deviation (RSD) was <2.0%. The present CL procedure was applied to the determination of VEN in pharmaceutical formulations and serum samples; the recovery levels were in the range 96.5-101.2%. The results suggest that the method is unaffected by the presence of common formulation excipients found in these samples.

New spectrofluorimetric method for determination of cephalosporins in pharmaceutical formulations.

A simple, accurate, precise spectrofluorimetric method has been proposed for the determination of three cephalosporins, namely, cefixime (cefi), cephalexine (ceph), and cefotaxime sodium (cefo) in pharmaceutical formulations. This method is based on a reaction between cephalosporins with 8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt (HPTS) in alkaline medium, at pH 12.0 for cefi and 13.0 for ceph and cefo to give highly fluorescent derivatives extracted with chloroform and subsequent measurements of the formed fluorescent products at 520, 500 and 510 nm after excitation at 480, 470 and 480 nm for cefi, ceph and cefo respectively. The optimum experimental conditions have been studied. Beer's law is obeyed over concentrations of 10-60 ng/mL, 5-35 ng/mL and 10-60 ng/mL for cefi, ceph and cefo, respectively. The detection limits were 4.20 ng/mL, 2.54 ng/mL and 4.09 ng/mL for cefi, ceph and cefo, respectively, with a linear regression correlation coefficient of 0.99783, 0.99705 and 0.9978 and recoveries in ranges 96.96-105.77, 96.13-102.55 and 95.45-105.39% for cefi, ceph and cefo, respectively. This method is simple and can be applied for the determination of cefi, ceph and cefo in pharmaceutical formulations in quality control laboratories.

Determination of meloxicam using europium sensitized luminescence in the presence of co-luminescence reagents.

A sensitive time-resolved luminescence method for the determination of meloxicam (MX) in methanol and in aqueous solution is described. The method is based on the luminescence sensitization of europium (Eu(3+)) by formation of ternary complex with MX in the presence of 1,10-phenanthroline as coligand, Tween-80 as surfactant and gadolinium ion as a co-luminescence reagent. The signal for Eu-MX-1,10-phenanthroline is monitored at λ(ex) = 360 nm and λ(em) = 620 nm. Optimum conditions for the formation of the complex in aqueous system were 0.01 M TRIS buffer, pH 8.0, 1,10-phenanthroline (6.0 × 10(-6) M), Gd(3+) (7.0 × 10(-6) M), Tween-80 (0.28%) and 1.75 mM of Eu(3+) which allows the determination of 20-800 ppb of MX with limit of detection (LOD) of 7 ppb. The relative standard deviations of the method range between 0.1 and 1.1% indicating excellent reproducibility of the method. The proposed method was successfully applied for the assay of MX in pharmaceutical formulations, plasma and in urine samples. Average recoveries of 99.8 ± 1.1%, 100.2 ± 0.9% and 100.9 ± 1.1% were obtained for MX in tablet, plasma and urine sample respectively.

Liquid chromatography-tandem mass spectroscopic method for the determination of zerumbone in human plasma and its application to pharmacokinetics.

A rapid, sensitive, specific and selective LC-MS/MS method for the determination of zerumbone (ZER) in human plasma using 2,4-diamino-6-(4-methoxyphenyl)-1,3,5-triazine (DMTZ) as an internal standard (IS) has been developed and validated. ZER was chromatographed on C8 column using a mobile phase of acetonitrile/water (80:20, v/v) at a flow rate of 0.25 ml min(-1) . Quantitation was achieved using ESI+ interface, employing multiple reaction monitoring (MRM) mode at m/z 219 > 81 and 218 > 134 for ZER and IS, respectively. The calibration standards were linear over a range of 5-3000 ng ml(-1) (r(2)=0.9994) with an LLOQ of 5 ng ml(-1) (RSD %; 11.4% and bias%; 9.5%). Intra- and inter-day precision of ZER assay ranged from 0.18 to 3.56% with accuracy (bias) that varied between -5.09 and 4.3%, demonstrating good precision and accuracy. Recoveries of ZER and the IS from human plasma were above 85%. The developed method was validated for the determination of ZER in rat plasma. Linearity, stability of ZER and the ME on rat plasma were discussed. The applicability of the developed method was demonstrated by measuring ZER in rat plasma samples following intravenous and intraperitoneal administration of ZER prepared in hydroxypropyl-ß-cyclodextrin (HPßCD) and sodium carboxymethyl cellulose (CMC), respectively, in 20 mg kg(-1) and this study indicated a clear significant difference (p<0.05) in pharmacokinetic parameters of ZER in ZER/HPßCD complex compared with ZER in CMC preparation.

High throughput method for the analysis of cetrizine hydrochloride in pharmaceutical formulations and in biological fluids using a tris(2,2'-bipyridyl)ruthenium(II)-peroxydisulphate chemiluminescence system in a two-chip device.

A fast, economic and sensitive chemiluminescence (CL) method has been developed for the analysis of cetrizine hydrochloride (CET) in pharmaceutical formulations and in biological fluids. The CL method is based on the oxidation of tris(2,2'-bipyridyl)ruthenium(II) (Ru (bipy)(3)(2+)) by peroxydisulphate in a two-chip device. Up to 180 samples can be analysed per hour, consuming only minute quantities of reagents. Three instrumental setups were tested to find the most economical, sensitive and high throughput setup. In the first setup, a continuous flow of sample and CL reagents was used, whereas in the second setup, a fixed volume (2 µL) of (Ru (bipy)(3)(2+)) was introduced into a continuous infusion of peroxydisulphate and the sample. In the third design, a fixed volume of sample (2 µL) was injected while the CL reagents were continuously infused. Compared to the first setup, a 200% signal enhancement was observed in the third setup. Various parameters that influence the CL signal intensity, including pH, flow rates and reagent concentrations, were optimized. A linear response was observed over the range of 50 µg L(-1) to 6400 µg L(-1) (R(2)=0.9959) with RSD values of 1.1% (n=15) for 1000 µg L(-1). The detection limit was found to be 15 µg L(-1) (S/N=3). The amount of consumed sample was only 2 µL, from which the detected amount of CET was found to be 6.5 × 10(-14)mol. This procedure was successfully applied to the analysis of CET in pharmaceutical formulations and biological fluids.

Analysis of fexofenadine in pharmaceutical formulations using tris(1,10-phenanthroline)-ruthenium(II) peroxydisulphate chemiluminescence system in a multichip device.

A simple, rapid and sensitive method has been developed for the analysis of fexofenadine (FEX) in pharmaceutical formulations, using a tris(1,10-phenanthroline)-ruthenium(II) [Ru(phen)(3)(2+)] peroxydisulphate chemiluminescence (CL) system in a multichip device. Various parameters that influence the CL signal intensity were optimized. These included pH, flow rates and concentration of reagents used. Under optimum conditions, a linear calibration curve in the range 0.05-5.0 µg/mL was obtained. The detection limit was found to be 0.001 µg/mL. The procedure was applied to the analysis of FEX in pharmaceutical products and was found to be free from interference from concomitants usually present in these preparations.

Spectrofluorimetric determination of aluminium using 2-hydroxy-1-naphthylidene-(8-aminoquinoline).

A sensitive and selective spectrofluorimetric method has been developed for the rapid determination of aluminium. This method is based on the complex formation between aluminium and 2-hydroxy-1-naphthylidene-(8-aminoquinoline) (HNAQ). The optimum conditions for the complex formation were a metal-to-ligand (M : L) stoichiometric ratio of 1:1, a pH of 5.5 and a 0.20 m acetate buffer. The fluorescence of the complex was monitored at an emission wavelength of 502 nm with excitation at 438 nm. Under these conditions, linear calibration curves were obtained in the ranges 0.05-1 and 1-5 ppm. The detection limit was 3.4 ppb for the former and 13.5 ppb for the latter. The maximum relative standard deviation of the method for an aluminium standard of 200 ppb was 1.5% (n = 5). This method was successfully applied for the determination of aluminium in drinking water, pharmaceutical antacid tablets and suspension samples.

Enhancement of on chip chemiluminescence signal intensity of tris(1,10-phenanthroline)-ruthenium(II) peroxydisulphate system for analysis of chlorpheniramine maleate in pharmaceutical formulations.

The effect of detection chip geometry on chemiluminescence (CL) signal intensity of tris(1,10-phenanthroline)-ruthenium(II) peroxydisulphate system for analysis of chlorpheniramine maleate (CPM) in pharmaceutical formulations was investigated. It was observed that the design of the detection chip is very crucial and can play an important role in enhancing the CL signal intensity in this system. The CL signal intensity was enhanced 250% when a teardrop micromixer chip was used, compared to the commonly used serpentine chip geometry. The study was conducted using a multi-chip device. In this device, chip 1 was used to prepare and pump the reagent mixture, whereas chip 3 was used for pumping the sample. The two chips were connected to the teardrop chip (2) via silica capillary where detection took place. Non-linear regression curve fitting of the calibration data revealed that the calibration curves are best described by third order polynomial equation with excellent correlation coefficients (R(2)=0.9998) for the concentration range 7.69 × 10(-8) to 5.12 ×1 0(-5)mol L(-1). A linear response is also observed over the range 7.69 × 10(-8) to 1.28 × 10(-5)mol L(-1) (R(2)=0.9996) and the detection limit was found to be 5.49 × 10(-8)mol L(-1). The device was successfully used for the analysis of CPM in tablets and a multi-component cough syrup. Results were reproducible with relative standard deviation (RSD) of 0.6-1.1%.

Chemiluminescence determination of chlorpheniramine using tris(1,10-phenanthroline)-ruthenium(II) peroxydisulphate system and sequential injection analysis.

A sequential injection (SI) method was developed for the determination of chlorpheniramine (CPA), based on the reaction of this drug with tris(1,10-phenanthroline)-ruthenium(II) [Ru(phen)(3)(2+)] and peroxydisulphate (S(2)O(8)(2-)) in the presence of light. The instrumental set-up utilized a syringe pump and a multiposition valve to aspirate the reagents [Ru(phen)(3)(2+) and S(2)O(8)(2-)] and a peristaltic pump to propel the sample. The experimental conditions affecting the chemiluminescence reaction were systematically optimized, using the univariate approach. Under the optimum conditions linear calibration curves of 0.1-10 microg/ml were obtained. The detection limit was 0.04 microg/ml and the relative standard deviation (RSD) was always < 5%. The procedure was applied to the analysis of CPA in pharmaceutical products and was found to be free from interferences from concomitants usually present in these preparations.

Determination of aminoglutethimide enantiomers in pharmaceutical formulations by capillary electrophoresis using methylated-beta-cyclodextrin as a chiral selector and computational calculation for their respective inclusion complexes.

A capillary electrophoretic method for the separation of the aminoglutethimide (AGT) enantiomers using methylated-beta-cyclodextrin (M-beta-CD) as chiral selector is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 9 min with resolution factor Rs=2.1, using a fused-silica capillary and a background electrolyte (BGE) of tris-phosphate buffer solution (50 mmol L(-1), pH 3.0) containing 30 mgm L(-1) of M-beta-CD. The separation was carried out in normal polarity mode at 25 degrees C, 16 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy/recovery were included. The proposed method was successfully applied to the assay of AGT enantiomers in pharmaceutical formulations. The computational calculations for the inclusion complexes of the R- and S-AGT-M-beta-CD rationalized the reasons for the different migration times between the AGT enantiomers.

Terbium sensitized luminescence for the determination of ketoprofen in pharmaceutical formulations.

This paper explores an ultra-sensitive luminescence method for the determination of Ketoprofen (KP) in pharmaceutical formulations. The technique is indirect and exploits the luminescence enhancement of terbium (Tb(3+)) by complexation with KP (Tb(3+)-KP), which was monitored at respective excitation and emission wavelengths of lambda(ex) = 258 nm and lambda(em) = 549 nm. The effect of varying the Tb(3+) concentration and using multiple solvents was examined to determine optimal experimental conditions. Maximum sensitization was accomplished in the presence of methanol where the most favourable condition for the formation of the complex was recorded at a level of 1.0 x 10(-5) M of Tb(3+). Under these optimum experimental conditions, linear calibration curve was obtained in the range of 2.8 x 10(-7)-3.1 x 10(-6) M with a detection limit of 8.7 x 10(-8) M. The technique was validated with 'working' reference standards and produced relative standard deviations < 2% indicating that the reproducibility was highly acceptable. The proposed method was successfully applied to assays of KP in pharmaceutical formulations with average recoveries of 92-98%. The results were found to be in good agreement with those obtained by HPLC. The method is highly suited for general applications of this nature.

A spectrofluorimetric sequential injection method for the determination of penicillamine using fluorescamine in the presence of beta-cyclodextrins.

A simple, robust and sensitive sequential injection spectrofluorimetric method for the determination of penicillamine (PA) in pharmaceutical formulations is developed. The method is based on the formation of a highly fluorescent derivative when penicillamine is reacted with fluorescamine (FL) in borate buffer of pH 9.3. The derivative produced is monitored at an emission wavelength of 495 nm using an excitation wavelength of 355 nm. The optimum conditions for the determination of PA with FL were: 3 mM FL, pH 9.3, 5 mM methyl-beta-cyclodextrin, sample volume of 75 microL and reagent volume of 75 microL. Furthermore, the effect of various media on the fluorescence intensity of the PA-FL derivative was studied and methyl-beta-cyclodextrin was found to give the largest enhancement. A linear dynamic range for the determination of PA of 5-80 ppm was obtained with a sampling frequency of 50 h(-1) and a relative standard deviation of less than 2.5%. The method was applied to the determination of PA in pharmaceutical formulations with reasonable recoveries ranging from 101.0-103.1%, indicating that no interference is observed from concomitants usually present in dosage forms.

Spectrofluorimetric determination of zinc using 8-hydroxy-7-(4-sulfo-1-naphthylazo)-5-quinoline sulfonic acid.

A sensitive and a selective spectrofluorimetric method have been developed for the rapid determination of zinc. The method is based on the complex formation between zinc and 8-hydroxy-7-(4-sulfo-1-naphthylazo)-5-quinoline sulfonic acid (HSNQ). The optimum conditions for the complex formation were metal to ligand stoichiometric ratio of 1:1 at pH 8.0 with 0.2M acetate buffer. The fluorescence of the complex is monitored at an emission wavelength of 545 nm with excitation at 360 nm. Under these conditions linear calibration curves were obtained from 50 ppb to 400 ppb. The detection limit was 7 ppb. The maximum relative standard deviation of the method was 2% for water samples, 4% for milk samples and 7% for hair samples (n = 5). The method was successfully applied for the determination of zinc in drinking water, hair and milk samples. The results were found to be in good agreement with those obtained by flame atomic absorption spectrophotometric methods. The t-test and F-test indicated no significant difference at 95% confidence level.

Enhancement of the chemiluminescence of penicillamine and ephedrine after derivatization with aldehydes using tris(bipyridyl)ruthenium(II) peroxydisulfate system and its analytical application.

A novel sequential injection (SI) method was developed for the determination of penicillamine (PA) and ephedrine (EP) based on the reaction of these drugs with tris(bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) and peroxydisulfate (S(2)O(8)(2-)) in the presence of light. Derivatization of PA and EP with aldehydes has resulted in a significant enhancement of the chemiluminescence emission signal by at least 25 times for PA and 12 times for EP, leading to better sensitivities and lower detection limits for both drugs. The instrumental setup utilized a syringe pump and a multiposition valve to aspirate the reagents, (Ru(bpy)(3)(2+) and S(2)O(8)(2-)), and a peristaltic pump to propel the sample. The experimental conditions affecting the derivatization reaction and the chemiluminescence reaction were systematically optimized using the univariate approach. Under the optimum conditions linear calibration curves between 0.2-24 microgmL(-1) for PA and 0.2-20 microgmL(-1) for EP were obtained. The detection limits were 0.1 microgmL(-1) for PA and 0.03 microgmL(-1) for EP. The procedure was applied to the analysis of PA and EP in pharmaceutical products and was found to be free from interferences from concomitants usually present in these preparations.

A sequential injection method for the fluorimetric determination of aluminum in drinking water using 8-hydroxy-7-(4-sulfo-1-naphthylazo)-5-quinoline sulfonic acid.

A robust and simple sequential injection (SI) method for the assay of aluminum ions in drinking water is described. The method is based on the complex formation between aluminum and 8-hydroxy-7-(4-sulfo-1-naphthylazo)-5-quinoline sulfonic acid (HSNQ). The fluorescence of the complex is monitored at an emission wavelength of 492 nm with excitation at 357 nm. The HSNQ concentration, aspirated reagent and sample volumes were optimized simultaneously using 3(3) full factorial design. The optimum operating conditions are aspirated sample and reagent volumes of 90 and 70 microL, respectively, and HSNQ concentration of 20 microM. With these conditions linear calibration curves were obtained from 100 to 800 ppb. The detection limit was 4 ppb. The maximum relative standard deviation of the method was 1.43% (n=5). The method was successfully applied for the determination of aluminum in drinking water samples.