Development of a solid-phase extraction method for determination of pheophorbide a and pyropheophorbide a in health foods by liquid chromatography

A simple solid-phase extraction (SPE) method was developed for the liquid chromatography (LC) determination of pheophorbide (Phor) a and pyropheophorbide (Pyro) a in health foods such as chlorella, spirulina, etc. The food sample was extracted with 85% (v/v) acetone. The extract was acidified with hydrochloric acid and loaded on a C18 cartridge. After washing with water, Phor a and Pyro a were eluted with the LC mobile phase. Phor a and Pyro a were separated by isocratic reversed-phase LC and quantitated by fluorescence detection. The recoveries for spiked samples of chlorella and the extract were 87.1-102.0%. Commercial health foods (chlorella, spirulina, aloe, kale, Jews mallow, and green tea leaves) were analyzed using the SPE method. The values found for Phor a and Pyro a ranged from 2 to 788 microg/g and from <1 to 24 microg/g, respectively. There was no significant difference between the SPE method and the official method in Japan (spectrophotometry after liquid-liquid extraction). The advantages of the SPE method are the short extraction times, lack of emulsions, and reduced consumption of organic solvents compared with the official method in Japan. The SPE method is considered to be useful for the screening of Phor a and Pyro a in health foods.     

Determination of niacin in infant formula by solid-phase extraction and anion-exchange liquid chromatography.

A peer-verified, solid-phase extraction (SPE)/anion exchange liquid chromatographic method is presented for the determination of niacin in milk-based and soy-based infant formula. Analysis is in 3 steps: test sample digestion, extraction/cleanup, and liquid chromatography (LC). Digestion uses a standard AOAC digestion procedure that involves autoclaving at 121 degrees C for 45 min in (1 + 1) H2SO4 to free endogenous niacin from protein and to convert added niacinamide to niacin. The digest solution is adjusted to pH 6.5 with 7.5M NaOH. Acidification to pH <1.0 with (1 + 1) H2SO4 precipitates the protein. The clarified solution is then filtered, and the filtrate is brought to volume. SPE of niacin is accomplished by passing an aliquot of the digest solution through an aromatic sulfonic acid-SPE (ArSCX-SPE) column. After the column is washed with methanol and water to remove extraneous material, the niacin is eluted with 0.25M sodium acetate/acetic acid buffer at pH 5.6. An anion-exchange polystyrene-divinylbenzene column with 0.1 M sodium acetate/acetic acid buffer at pH 4.0 is used for LC. Niacin is determined by UV detection at 260 nm. A standard curve is prepared by passing known amounts of niacin through the ArSCX-SPE columns used for niacin extraction. The following values for x and relative standard deviation (RSD) were obtained for National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1846 Infant Formula with a certified value for niacin of 63.3 +/- 7.6 microg/g: Submitting laboratory.-- x = 59.7 +/- 4.0 microg/g; RSD = >6.7%; confidence interval (CI) = +/- 1.4 microg/g; n = 27. Peer laboratory.--x = 56.6 +/- 6.6 microg/g; RSD = >11.7%; CI =+/- 4.1 microg/g; n = 8.     

Determination of seventeen polar/thermolabile pesticides in apples and apricots by liquid chromatography/mass spectrometry

A simple liquid chromatography/mass spectrometry (LC/MS) approach for the determination of widely used representatives of polar/thermolabile pesticides in fruits was developed and validated. The group of pesticides comprised benzimidazoles and azoles (carbendazim, thiabendazole, imazalil, propiconazole, prochloraz, epoxiconazole, flusilazole, tebuconazole, bitertanol); N-methylcarbamates (carbaryl, carbofuran, methiocarb); and phenylureas and benzoylphenylureas (linuron, diflubenzuron, triflumuron, teflubenzuron, flufenoxuron). Matrixes (apple, apricot) were extracted with acetonitrile and crude extracts were cleaned up by solid-phase extraction (SPE) using either mixed cation exchange or hydrophilic lipophilic balance cartridges. LC separation of pesticides was performed on a reversed-phase column, Discovery C18. Electrospray ionization and ion trap MS/MS detection were applied. For most pesticides, overall recoveries ranged from 75 to 122%, and repeatability (as relative standard deviation) from 5 repetitive determinations of recovery ranged from 3 to 21%. Carbofuran was the only compound for which recovery was not satisfactory due to its loss in the SPE cleanup step. Limits of detection were 0.1-3 microg/kg for benzimidazole and azole fungicides and carbamate insecticides. For urea insecticides, detection limits were slightly higher (3-10 microg/kg).     

Determination of melamine in pet food by enzyme immunoassay, high-performance liquid chromatography with diode array detection, and ultra-performance liquid chromatography with tandem mass spectrometry

Melamine in pet food (fortified or originally contaminated) was determined by enzyme immunoassay (EIA), high-performance liquid chromatography with diode array detection (HPLC-DAD), and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The limits of detection (LOD) for EIA and HPLC-DAD were 0.02 and 0.1 microg/mL, respectively. The linear ranges of the calibration curves for EIA and HPLC-DAD were 0.02-0.5 and 0.1-500 microg/mL, respectively. The coefficient of determinations (r2) of the standard curves for EIA and HPLC were 0.9991 and 0.9999, respectively. Coefficient of variations from both inter- and intra-assay were <9.31%, and recovery range for all concentrations was between 71 and 105%. The r2 values between the EIA and HPLC-DAD methods for melamine analysis of the fortified and originally contaminated samples were 0.9973 and 0.9885. The r2 values for UPLC-MS/MS with HPLC-DAD and with EIA were 0.9566 and 0.9489, respectively.     

Determination of halofuginone and amprolium in chicken muscle and egg by liquid chromatography

A liquid chromatographic (LC) method was developed for simultaneous measurement of halofuginone (HFN) and amprolium (APL) in chicken muscle and egg. HFN and APL were extracted from chicken muscle and egg with acetonitrile. In chicken egg, they were partially purified by solid-phase extraction (SPE) to separate them from impurities. The LC separation was performed on a 4.6 mm id x 250 mm TSK-gel ODS-80TM column using acetonitrile-McIlvaine buffer, pH 3.4, containing 0.01M sodium lauryl sulfate (42 + 58) as the mobile phase. Ultraviolet detection of HFN and APL was performed at wavelengths of 242 and 265 nm, respectively. Recoveries of HFN and APL from chicken muscle spiked at 0.5 microg/g were 74.8 +/- 17.7 and 94.2 +/- 5.0%, respectively (mean +/- standard deviation [SD], n = 10). In chicken muscle, the lower limit of determination for both APL and HFN was 0.03 microg/g. Recoveries of HFN and APL from chicken egg spiked at 0.5 microg/g by a cleanup procedure using SPE were 54.6 +/- 3.4 and 85.0 +/- 2.4%, respectively (mean +/- SD, n = 5). In chicken egg, the lower limit of determination for both APL and HFN was 0.04 microg/g.     

Sample preparation for determination of macrocyclic lactone mycotoxins in fish tissue, based on on-line matrix solid-phase dispersion and solid-phase extraction cleanup followed by liquid chromatography/tandem mass spectrometry

A new method based on matrix solid-phase dispersion (MSPD) on-line with a solid-phase extraction (SPE) cleanup process followed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) is presented for the determination of 3 macrocyclic lactone mycotoxins in fish tissues: zearalenone, alpha-zearalenol, and beta-zearalenol. The sample was prepared in a device that used a reversed-phase material (C18) or a normal-phase material (neutral alumina) as a matrix dispersing agent, and a graphitized carbon black cartridge was used for sequential cleanup by SPE. LC/MS/MS was used for selective determination. Isocratic elution with acetonitrile-methanol-water was used for LC separation; for MS/MS, 2 types of interfaces (a pneumatically assisted electrospray ionization interface or an atmospheric pressure chemical ionization interface) were evaluated and compared in terms of the intensity of the total ion current produced by each analyte. The use of highly selective MSPD on-line with SPE for sample preparation before analysis allowed the removal of interfering matrix compounds present in tissue extracts that would otherwise cause severe ionization suppression of zearalenone and its metabolites during the ionization process. Average recoveries at 100 ng/g were between 83 and 103% with C18 and > or = 67% with neutral alumina; the relative standard deviations were < 11% with C18 and < 18% with alumina. The limits of detection ranged from 0.1 to 1.0 ng/g. Sample preparation is simple to perform, no special technical equipment is required, and solvent volumes are minimal.     

HPLC method for the determination and pharmacokinetic studies on geniposide in rat serum after oral administration of traditional Chinese medicinal preparation Yin-Zhi-Ku decoction

A new HPLC method for the determination of geniposide in rat serum with solid-phase extraction (SPE) for preconcentration is described. Geniposide and an internal standard (paeoniflorin) were extracted from serum by SPE using C18 cartridges. Analysis of the extract was then performed on a reversed-phase C18 column using acetonitrile-water (16:84, v/v) as the eluting solvent system, and UV detection at 238 nm was used to measure the analyte with a limit of quantitation about 0.1 microg/mL. The calibration curve for geniposide was linear (r = 0.9993) in the concentration range 0.1-16.0 microg/mL. The intra- and inter-day precision of the geniposide were determined and their RSD did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of geniposide from rat serum after oral administration of Yin-Zhi-Ku decoction.     

气相色谱-质谱法同时测定化妆品中19种邻苯二甲酸酯

建立了同时测定化妆品中19种邻苯二甲酸酯的气相色谱-质谱(GC-MS)方法。样品经二氯甲烷超声提取后,用硅胶-中性氧化铝(2:3, m/m)混合填料固相小柱保留、乙酸乙酯-正己烷(8:2, v/v)洗脱等步骤净化后,采用GC-MS对19种目标物进行定性、定量分析。采用保留时间与特征离子丰度比共同进行定性分析,减少了复杂样品基质中的干扰。在优化的实验条件及0.1、0.5、2.0 μg/g的添加水平下,平均加标回收率为72.2%～110.9%,其中加标水平为0.1 μg/g的相对标准偏差(RSD, n=6)小于10.3%;方法检出限(以3倍标准偏差计)为0.0065 μg/g(邻苯二甲酸二异戊酯)～0.062 μg/g(邻苯二甲酸二异丁酯)之间。应用该方法检测了6种化妆品中19种PAEs的含量。该方法可推广用于多种类型化妆品中PAEs的测定。     

Rapid determination of nitroimidazole residues in honey by liquid chromatography/tandem mass spectrometry

We developed a rapid and efficient means of determining residues of four nitroimidazoles-i.e., dimetridazole, ipronidazole, metronidazole, and ronidazole-and three hydrophilic metabolites- i.e., 2-hydroxymethyl-1-methyl-5-nitroimidazole, 1 -methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole, and 1-(2-hydroxyethyl)-2-hydroxymethyl-nitroimidazole--in honey. We applied a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure improved to suit a nitroimidazole analysis, which is fast (approximately 30 min) and uses less organic solvent. The procedure involves initial single-phase extraction of 5 g of honey with acetonitrile containing 1% acetic acid, followed by liquid-liquid partitioning involving the addition of 5 g sodium chloride, 1.5 g trisodium citrate dihydrate, and 4 g magnesium sulfate. Moreover, matrix from honey was reduced by an SPE method with an alumina-N cartridge. The samples were analyzed using LC/MS/MS. Chromatographic separation of these nitroimidazoles and metabolites was performed in the gradient mode on a pentafluorophenylpropyl-bonded silica column (150x2.0 mm, 3 pm particle size) at 40 degrees C. The mobile phase consisted of a 0.01% acetic acid solution and acetonitrile, and the flow rate was 0.2 mL/min. The method was validated using honey spiked with these nitroimidazoles from 0.1 to 0.5 microg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were <9.5 and <14.2%, respectively. The LOQ ranged from 0.1 to 0.5 microg/kg. LC/MS/MS coupled with the QuEChERS method showed good potential as a method for determining nitroimidazole residues in honey.     

Determination of fluorescent whitening agents in laundry detergents and surface waters by solid-phase extraction and ion-pair high-performance liquid chromatography

A simple method was developed to detect four stilbene-type disulfonate and one distyrylbiphenyl-type fluorescent whitening agents (FWAs) in household laundry detergents and surface waters by ion-pair high-performance liquid chromatography. The FWA concentrations in detergents were measured directly. The contents of FWAs in water samples were extracted by solid-phase extraction (C18-SPE) with ion-pairing reagent, and were then determined by an isocratic ion-pair chromatography (IPC) using a C18 column, applying tetrabutylammonium hydrogensulfate (TBA) as the ion-pairing reagent in mobile phase, and equipped with fluorescence detection. Water samples at various pH conditions for SPE were evaluated. Experimental results indicate that the proposed method is precise and sensitive in analyzing FWAs, and enables quantitation of 0.01-0.1 microg/l in 100 ml water samples. The recovery rates of FWAs in spiked water samples were between 73 and 89%, and the precision (RSD) ranged from 2.6 to 8.9%. Over 7200 microg/g of 4,4'-bis(2-sulfostryl)-biphenyl (DSBP) and 2320 microg/g of 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazine-2-yl)-amino]stilbene-2,2'-disulfonate (DAS1) were detected in household laundry detergents. Trace amounts of DSBP were detected in surface water samples ranging from 0.2 to 3.7 microg/l.     

Comparison of different liquid chromatography methods for the determination of corticosteroids in biological matrices

Various extraction techniques can be combined with column liquid chromatography (LC) and ultraviolet (UV) or mass spectrometric (MS) detection for the determination of synthetic corticosteroids in biological matrices. Target analysis of low concentrations of 25 microg/kg of dexamethasone in feed can be performed by combining immunoaffinity chromatography (IAC) and LC with UV detection. A straightforward multi-analyte procedure is obtained by tandem solid-phase extraction (SPE) and subsequent LC-UV. However, the limits of detection for feed samples are then relatively poor, viz. 100 microg/kg. A multi-analyte method which meets modern demands of about 5 microg/kg detection limit requires one-step SPE combined with LC-MS analysis. As regards urine corticosteroids can be determined down to a level of 0.5 microg/l by either SPE-LC-MS- MS or SPE(IAC)-LC-MS.     

New fluorimetric method of liquid chromatography for the determination of the neurotoxin domoic acid in seafood and marine phytoplankton

Domoic acid (DA) is a neurotoxic amino acid that is responsible for the human toxic syndrome, amnesic shellfish poisoning (ASP). A new rapid, sensitive liquid chromatographic (LC) method has been developed for the determination of DA in various marine samples. DA in marine biological materials was derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and analysed using isocratic reversed-phase LC with fluorimetric detection. The calibration, based on standard DA solutions, was linear in the range 0.04-2 microg/ml (r2=0.998) and the detection limit (3:1, signal/noise) was better than 1 ng/ml. Using the certified reference material (MUS-1B), recoveries of DA from shellfish tissue were >95% (n=5). When a strong anion exchange SPE cartridge was used for sample clean-up the detection limit was 6 ng DA/g mussel tissue. Good reproducibility was achieved with RSD values ranging from 3% for 8 microg DA/g (n=5), to 5% for 0.04 microg DA/g (n=5). This new method was successfully applied to the determination of DA in naturally contaminated shellfish and in marine phytoplankton cultures of Pseudonitzschia sp.     

Determination of microcystins in fish by solvent extraction and liquid chromatography

A liquid chromatography electrospray mass spectrometry (LC/ESI/MS) method has been developed to identify and quantify microcystins in fish liver and intestine. Microcystins (MCs) were extracted from 500 mg sample with methanol-water (85:25, v/v) and the extracts concentrated to 250 microl. The parameters were optimized by a full factorial 2(3) design. Neither laborious pre-treatment nor clean up were necessary. MCs were separated using conventional C18 column and an acetonitrile-acidified water (pH 3) gradient. Negative samples (without MCs) were discriminated by liquid chromatography diode array detection (LC/DAD). The limits of detection (LOD) and the limits of quantification (LOQ) resulted equal for MC-RR, MC-YR, and MC-LR and were 0.1 and 0.5 microg g(-1), respectively. MCs recoveries at three levels in spiked samples (0.5-3.0 microg g(-1)) were > 92%, with relative standards deviations (RSDs) < 16% for liver samples and > 68% with RSDs < 18% for intestine samples. The proposed method was applied to determine MC-LR in exposed fish to evaluate the bioaccumulation risk. The results showed the transference of MC-LR from cyanobacterial cells to fish tissues.     

Determination of tetrodotoxin in puffer-fish by liquid chromatography-electrospray ionization mass spectrometry

A simple and reliable method using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for the determination of tetrodotoxin in the puffer-fish has been developed. The LC separation was performed on a Shodex RSpak NN-414 column (15 cm x 4.6 mm id) using 20 mM ammonium acetate-methanol (75 + 25) as the mobile phase at a flow rate of 0.5 ml min(-1). The positive ionization produced the typical [M + H]+ molecular ion of tetrodotoxin (m/z 320). The calibration graph for tetrodotoxin was rectilinear from 0.01 to 1 microg ml(-1) with selected ion monitoring (SIM). Tetrodotoxin was extracted with 0.1% acetic acid by heating in a boiling water-bath and the extracts were cleaned up on a Bond Elut C18 (500 mg) cartridge. The recoveries of the tetrodotoxin from the puffer-fish fortified at 1 microg g(-1) were 77.7-80.7% and the detection limit was 0.1 microg g(-1) (equivalent to ca. 0.5 mouse units per gram).     

Simultaneous determination of 16 sulfonamides in honey by liquid chromatography/tandem mass spectrometry

A method is described for the determination of 16 sulfonamides in honey. Samples are dissolved in phosphoric acid solution (pH2), cleaned up with 2 solid-phase extraction (SPE) cartridges, an aromatic sulfonic cation-exchange cartridge and an Oasis HLB SPE cartridge, and analyzed both qualitatively and quantitatively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) under the selected conditions. Without exception, calibration curves were linear (r = > 0.995), when sulfamethizole was between 1.0 and 25.0 microg/kg; sulfacetamide, sulfapyridine, sulfadiazine, sulfachloropyridazine, sulfamethoxazole, sulfamerazine, sulfisoxazole, sulfamonomethoxine, and sulfadoxine were between 2.0 and 50.0 microg/kg; sulfamethoxypyridazine, sulfadimethoxine, and sulfathiazole were between 4.0 and 100.0 microg/kg; sulfamethazine and sulfameter were between 8.0 and 200.0 microg/kg; and sulfaphenazole was between 12.0 and 300.0 microg/kg. Average recoveries at 4 fortification levels in the range of 1.0-300 microg/kg in honey were 70.9-102.5%, and relative standard deviations were 2.02-11.52%. The limits of quantitation for the 16 sulfonamides were between 1.0 and 12.0 microg/kg, with the LC/MS/MS method.     

Analyses of pesticide residues in fruit-based baby food by liquid chromatography/electrospray ionization time-of-flight mass spectrometry

A liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) method has been developed for the determination of 12 pesticides (namely, carbendazim, thiabendazole, imazalil, tridemorph, triadimefon, bitertanol, prochloraz, flutriafol, myclobutanil, iprodione, diphenylamine and procymidone) in fruit-based baby food (multi-fruit jars and juices intended for infant consumption). The developed method consists of a sample treatment step based on liquid-liquid extraction using acetonitrile, followed by a clean-up step based on dispersive solid-phase extraction (SPE) with a primary-secondary amine (PSA). Multi-fruit and apple juices were processed by a SPE procedure using Oasis HLB cartridges. Subsequent identification and quantitation was accomplished by LC/ESI-TOFMS analysis: the confirmation of the target pesticides was based on accurate mass measurements of selected ions (protonated molecules ([M+H]+) and fragment ions). Confirmation studies were accomplished at low concentration levels (10 microg kg-1) and accuracy errors lower than 2 ppm were obtained in most cases. Baby food extracts spiked at 10 microg kg-1 fortification level yielded average recoveries in the range 78-105% with relative standard deviations less than 10% for most of the analytes. Limits of detection (LODs) were between 0.1 and 4 microg kg-1 depending on the pesticide studied. Finally, the proposed method was applied to a total of 33 baby food samples from Spain and the United Kingdom. Although imazalil, thiabendazole and carbendazim were detected in a high number--over 60%- of baby food samples, none of the samples tested were found to be above the 0.01 mg kg-1 EU standard.     

Determination of benzene, toluene, ethylbenzene and xylene in river water by solid-phase extraction and gas chromatography.

A rapid and reproducible method is described that employs solid-phase extraction (SPE) using dichloromethane, followed by gas chromatography (GC) with flame ionization detection for the determination of benzene, toluene, ethylbenzene, xylene and cumene (BTEXC) from Buriganga River water of Bangladesh. The method was applied to detect BTEXC in a sample collected from the surface, or 5 cm depth of water. Two-hundred milliliters of n-hexane-pretreated and filtered water samples were applied directly to a C18 SPE column. BTEXC were extracted with dichloromethane and the BTEX concentrations were obtained to be 0.1 to 0.37 microg ml(-1). The highest concentration of benzene was found as 0.37 microg ml(-1) with a relative standard deviation (RSD) of 6.2%; cumene was not detected. The factors influencing SPE e.g., adsorbent types, sample load volume, eluting solvent, headspace and temperatures, were investigated. A cartridge containing a C18 adsorbent and using dichloromethane gave a better performance for the extraction of BTEXC from water. Average recoveries exceeding 90% could be achieved for cumene at 4 degrees C with a 2.7% RSD.     

Determination of quinolones in chicken tissues by liquid chromatography with ultraviolet absorbance detection

This paper presents an analytical method for the determination of quinolones in chicken tissues. The procedure involves pre-treatment by solid-phase extraction (SPE) and subsequent liquid chromatography (LC) with UV absorbance detection. Different SPE disposable cartridges and extractants of the tissue samples were tested, and various columns were systematically tested. The mobile phase was composed of acetonitrile and citric buffer at pH 4.5, with an initial composition of acetonitrile-water (12:88, v/v) and using linear gradient elution. Recoveries were 66-91% in the concentration range 30-300 microg kg(-1). The detector response was linear in this range. The limits of detection were 16-30 microg kg(-1). These values were lower than the maximum residue limits established by the European Union.     

Determination of proline enantiomers in honey and royal jelly by LC-UV

The chiral separation and quantification of D-proline and L-proline in honey and royal jelly were examined by LC with UV detection. Most of the endogenous compounds existing in honey, such as sugars, were removed by using SPE cartridges containing C18 and strong cation-exchange sorbent. Other components, such as primary amino acids, were also removed by two-step derivatization with o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC-CI). The components that were derivatized with OPA were separated from proline with a C18 cartridge. Proline was then converted into an FMOC derivative that could be subsequently measured by LC-UV. Sufficient chiral separation of D-proline and L-proline was achieved with an LC chiral column made of a beta-cyclodextrin phase in the polar organic-phase mode. The average recoveries of D-proline and L-proline from honey and royal jelly were in the range of 81.3-98.6% (RSD of < 1.8%). When this method was applied to commercial honey and royal jelly samples, L-proline was detected at concentrations of 369-1930 microg/g, whereas D-proline was not detected.     

Determination of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urine by high-performance liquid chromatography combined with mass spectrometry

A simple, sensitive and specific method for the quantification of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urines of the general population and of exposed workers has been developed. Samples were first diluted with phosphate buffer followed by purification by solid-phase extraction (SPE) with SAX columns prior to analysis by liquid chromatography/mass spectrometry (LC/MS). An external standard was used for the quantification together with selected ion monitoring of m/z 219, [M-H]-. The linear calibration curve showed a good correlation (r2 = 0.98, p < 0.001) between 0.1-110 mg/L of AMCC, and the detection limit was calculated to be 2 microg/L. The within-day and between-day precision, calculated for exposed workers and general population AMCC levels in urine samples, were determined as 1.2 and 3.6%, respectively (as relative standard deviation (RSD)).