Direct analysis of reference biofluids by coupled in situ electrodeposition-electrothermal atomic absorption spectrometry

The application of coupled in situ electrodeposition-electrothermal atomic absorption spectrometry (ED-ETAAS) to the determination of Pb in biological standard reference materials is described. In situ electrodeposition at a cell voltage of 3.0 V from 25-μl samples onto electrodeposited Pd is used to quantitatively separate the analyte from blood and urine matrices. With subsequent withdrawal of spent electrolyte, this overcomes the atomisation problems inherent with high salt and organic contents. ED-ETAAS is applied with minimal sample pre-treatment (acidification). The electrolysis process aids decomposition of the organic matrix, and the release of trace elements. Evolution of H₂ at the cathode counters fouling of the Pd modifier surface. The palladium deposit is renewed in situ for each determination. For AMI certified lyophilised blood, diluted 1+3 with 0.1 M HCl (18.1 μg/l Pb), the R.S.D. was 3.0% (peak height; n=5) and the detection limit (3 σ_blank; n=5) was 1.5 μg/l. Results for certified blood samples were AMI 72.3±4.3 μg/l (certified 76.2±7.6 μg/l) and Seronorm 34.2±2.0 μg/l (36±4 μg/l). The result for NIST SRM 2670 normal urine acidified to 1% HNO₃ was 8.1±0.6 μg/l (recommended value 10 μg/l).     

Mass spectrometric confirmation criterion for product-ion spectra generated in flow-injection analysis. Environmental application

The suitability of a confirmation criterion recently recommended in the Netherlands for gas chromatography with mass spectrometric detection (GC–MS), was evaluated for flow-injection analysis (FIA) with atmospheric pressure chemical ionisation MS–MS detection. The main feature of the criterion is that the relative ion abundances of the four diagnostic ions are taken into account. That is, for lower-intensity peaks, relative standard deviations may be higher; this is an advantage with chemical ionisation MS procedures. A series of triazines and their degradation products were used as test compounds. Tap and surface water samples spiked at 0.33 μg/l were analysed by means of a selected reaction monitoring MS–MS procedure. For all analytes but hydroxysimazine (3 transitions), 4–9 transitions could be selected which invariably met the demands of the criterion. Some of the transitions used originate from the ³⁷Cl isotopic mass of the parent compounds which provides additional structural information. Data for twenty surface water samples analysed by means of FIA–MS–MS as well as GC–MS and liquid chromatography with diode array UV and MS–MS detection gave essentially the same results over the 0.1–1.0 μg/l range. In two samples desethylatrazine was reported by FIA–MS–MS whereas this compound was not detected by GC–MS. For a first test, this is a promising result.     

Automated at-line solid-phase extraction-gas chromatographic analysis of micropollutants in water using the PrepStation

A fully automated at-line solid-phase extraction-gas chromatography procedure has been developed for the analysis of aqueous samples using the PrepStation. The sample extract is transferred from the sample preparation module to the gas chromatograph via an autosampler vial. With flame-ionization detection, limits of determination (S/N=10) of 0.05–0.13 μg/l were obtained for the analysis of HPLC-grade water when modifying the PrepStation by: (i) increasing the sample volume to 50 ml, (ii) increasing the injection volume up to 50 μl, and (iii) decreasing the desorption volume to 300 μl. The HP autosampler had to be modified to enable the automated “at-once” on-column injection of up to 50 μl of sample extract. The amount of packing material in the original cartridge had to be reduced to effect the decrease of the desorption volume. The total set-up did not require any further optimization after having set up the method once. The analytical characteristics of the organonitrogen and organophosphorus test analytes, i.e. recoveries (typically 75–105%), repeatability (2–8%) and linearity (0.09–3.0 μg/l) were satisfactory. The potential of the system was demonstrated by determining triazines and organophosphorus pesticides in river Rhine water at the 0.6 μg/l level using flame-ionization and mass-selective detection. No practical problems were observed during the analysis of more than 100 river water samples.     

Liquid chromatography — Particle beam mass spectrometry for identification of unknown pollutants in water

Summary Trace enrichment on a precolumn packed with copolymer material, coupled on-line with reversed-phase, column liquid chromatography-particle beammass spectrometry (RPLC-PB-MS) has been used for both target and non-target analysis of water samples. RPLC is carried out on a C-18-bonded silica column using a linear acetonitrile-0.1 M ammonium acetate gradient. Using optimised PB-MS conditions and 100–250 ml water samples, the detection limits for several phenylureas are in the 0.03–0.05 g l^–1 range using the full-scan mode; repeatability is good and the LC-PB-MS system is robust. Several surface and drinking water samples have been analysed and low levels of various environmental contaminants have been identified using electron impact mass spectra. Applying chemical ionisation with methane as reagent gas in both the positive and negative mode in conjunction with PB-MS provides relevant confirmatory information.     

Solid-phase extraction of polar pesticides from environmental water samples on graphitized carbon and Empore-activated carbon disks and on-line coupling to octadecyl-bonded silica analytical columns

The suitability of Empore-activated carbon disks (EACD), Envi-Carb graphitized carbon black (GCB) and CPP-50 graphitized carbon for the trace enrichment of polar pesticides from water samples was studied by means of off-line and on-line solid-phase extraction (SPE). In the off-line procedure, 0.5-2 1 samples spiked with a test mixture of oxamyl, methomyl and aldicarb sulfoxide were enriched on EnviCarb SPE cartridges or 47 mm diameter EACD and eluted with dichloromethane-methanol. After evaporation, a sample was injected onto a C₁₈-bonded silica column and analysed by liquid chromatography with ultraviolet (LC-UV) detection. EACD performed better than EnviCarb cartridges in terms of breakthrough volumes (>2 1 for all test analytes), reproducibility (R.S.D. of recoveries, 4–8%, n=3) and smapling speed (100 ml/min); detection limits in drinking water were 0.05–0.16 μg/l. In the on-line experiments, 4.6 mm diameter pieces cut from original EACD and stacked onto each other in a 9 mm long precolumn, and EnviCarb and CPP-50 packed in 10×2.0 mm I.D. precolumn, were tested, and 50–200 ml spiked water samples were preconcentrated. Because of the peak broadening caused by the strong sorption of the analytes on carbon, the carbon-packed precolumns were eluted by a separate stream of 0.1 ml/min acetonitrile which was mixed with the gradient LC eluent in front of the C₁₈ analytical column. The final on-line procedure was also applied for the less polar propoxur, carbaryl and methiocarb. EnviCarb could not be used due to its poor pressure resistance. CPP-50 provided less peak broadening than EACD: peak widths were 0.1–0.3 min and R.S.D. of peak heights 4–14% (n = 3). In terms of analyte trapping efficiency on-line SPE-LC-UV with a CPP-50 precolumn also showed better performance than when Bondesil C₁₈/OH or polymeric PLRP-S was used, but chromatographic resolution was similar. With the CPP-50-based system, detection limits of the test compounds were 0.05–1 μg/l in surface water.     

Evaluation of an on-line coupled continuous flow liquid membrane extraction and precolumn system as trace enrichment technique by liquid chromatographic determination of bisphenol A

An on-line coupled continuous flow liquid membrane extraction (CFLME) and C₁₈ precolumn system was developed for sample preconcentration in liquid chromatography determination. After preconcentration by CFLME, which is based on the combination of continuous flow liquid–liquid extraction and supported liquid membrane, bisphenol A (BPA) was enriched in 960 μl of 1 mol l⁻¹ NaOH used as acceptor. This acceptor was on-line neutralized and transported onto the C₁₈ precolumn where analytes were absorbed and focused. Then the focused analytes were injected onto a C₁₈ analytical column for separation and detected at 220 nm with a diode array detector. CFLME related parameters such as flow rates, pH of donor and acceptor, and enrichment time were optimized. The proposed method presents a detection limit of 0.03 μg l⁻¹ (S/N=3) when 60 ml samples was enriched with an enrichment time of 30 min. Compared with C₁₈ based column-switching procedure, this proposed procedure presents similar sample throughput and lower detection limits. The proposed method was successfully applied to determine BPA in tap water, river water, and municipal sewage effluent samples.     

On-line liquid chromatography-electrospray ionization mass spectrometry for determination of the brevetoxin profile in natural “red tide” algae blooms

Reversed-phase liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was employed to analyze brevetoxin compounds associated with naturally occurring red tide blooms collected from Sarasota Bay, FL, USA. The LC-ESI-MS method utilizes a C₁₈ microbore column with a mobile phase consisting of methanol-water (85:15, v/v), a flow-rate of 8 μl/min and a post-column split ratio of 3:1 (UV-absorbance detector-mass spectrometer). Three known brevetoxins Btx-2, Btx-1 and Btx-3 were detected at 60 μg/l, 10 μg/l and 5.7 μg/l levels, respectively, in the natrual red tide bloom samples. This distribution differed quantitatively from that found in red tide culture extract samples. Btx-9 was not detected either in natural red tide bloom extracts or in red tide culture extracts, possibly due to the instability of this compound. An unknown component with a molecular mass of 941 found in the natural bloom extract was postulated to have the structure of a reduced form (hydrogenation of two double bonds in the alkyl side chain) of Btx-5.     

On-line flow injection solvent extraction for electrothermal atomic absorption spectrometry: Determination of nickel in biological samples

A flow injection (FI) on-line solvent extraction system for electrothermal atomic absorption spectrometry (ETAAS) was developed with nickel as a model trace element. The nickel pyrrolidine-dithiocarbamate chelate was extracted on line into isobutyl methyl ketone, which was delivered into the FI system by a peristaltic pump equipped with poly(tetrafluoroethylene) tubing. The organic phase was separated from the aqueous phase by a novel gravity phase separator with a small conical cavity, and stored in a collector tube, from which 50 μl organic concentrate was introduced into the graphite tube by an air flow. ETAAS determination of the analyte was performed in parallel with the extraction process. An enrichment factor of 25 was obtained in comparison with 50 μl direct introduction while achieving a detection limit of 4 ng l⁻¹ (3σ), and a precision of 1.5% relative standard deviation for 1.0 μg l⁻¹ nickel (n = 11). The proposed method was successfully applied to the determination of nickel in body fluids and other biological samples.     

Determination of fenpyroximate in apples by supercritical fluid extraction and packed capillary liquid chromatography with UV detection

A method using off-line supercritical fluid extraction (SFE) and micro liquid chromatography (μLC) with UV detection at 260 nm, was developed for selective determination of fenpyroximate in apple samples. The packed capillary liquid chromatography method utilises 20 μl injection volumes with on-column focusing. A 350×0.32 mm capillary column packed with Kromasil 100-C₁₈ of 5 μm particle size was used with a mobile phase of acetonitrile–10 mM ammonium acetate (85:15, v/v) at a flow of 5 μl/min. A two-step SFE procedure was used to extract fenpyroximate selectively in 2 g apple samples, with Hydromatrix (HMX) added as a water absorbent at a 1:1 (w:w) ratio. Fenpyroximate was extracted at 200 bar and 90°C for 15 min using carbon dioxide at a flow of 2 ml/min, and solvent trapping collection in 10 ml acetonitrile. The volume of the acetonitrile extract was reduced by evaporation and water was added to a final composition of acetonitrile–water (40:60, v/v). The resulting 2.0 ml solution was filtered using a 0.45 μm poly(vinylidene difluoride) syringe filter before μLC analysis. Validation of the method was accomplished with apple samples spiked with fenpyroximate, covering the range of 0.1 to 1.0 μg/kg. The within-day and between-day repeatabilities were in the range 4–18% relative standard deviation. Accuracy, measured as recovery, was found to be approximately 60%. Apple samples from a field treated with fenpyroximate were analysed. None of the samples contained fenpyroximate above the quantification level.     

基于通过型固相萃取-超高效液相色谱-串联质谱法同时测定牛蛙中9种雌激素北大核心CSCD

建立了基于通过型固相萃取小柱净化的超高效液相色谱-三重四极杆质谱联用(UPLC-MS/MS)同时快速准确测定牛蛙中9种雌激素(雌三醇(E3)、17β-雌二醇(β-E)、17α-雌二醇(α-E)、17α-炔二雌醇(EE2)、雌酮(EI)、己烯雌酚(DES)、己二烯雌酚(DE)、己烷雌酚(HEX)、醋酸双烯雌酚(DD))残留的检测方法。样品经乙腈提取,经PRiME HLB固相萃取柱净化,Waters Acquity UPLC BEH C_(18)柱(100 mm×2.1 mm,1.7μm)分离,以0.5 mmol/L氟化铵水溶液-乙腈体系为流动相梯度洗脱,流速为0.3 mL/min,采用电喷雾正负离子切换模式(ESI^(+)/ESI^(-))和多反应监测(MRM)扫描方式检测,基质匹配外标法定量分析。该研究优化了液相色谱条件,相比于乙酸铵水溶液-乙腈体系和氨水溶液-乙腈体系,0.5 mmol/L氟化铵水溶液-乙腈体系作为流动相时9种雌激素普遍具有更佳的灵敏度。相比于甲醇和乙酸乙酯,乙腈作为提取溶剂时9种雌激素的提取率提高15%~40%。考察了HLB、C_(18)、Silica、PRiME HLB共4种不同类型的固相萃取小柱的基质净化效应,结果表明,PRiME HLB柱具有更好的基质净化能力。经PRiME HLB净化后,所有化合物的回收率均在70%~125%之间。DD的回收率从47%提高到74%,DES的回收率从180%降低到123%,有效减弱了基质效应。在最佳的实验条件下,E3、β-E、α-E、EI、DE、HEX、DD的线性范围为0.5~100.0μg/L,EE2和DES的线性范围为1.0~100.0μg/L,9种雌激素在各自的线性范围内均有良好的线性关系,相关系数为0.9953~0.9994,方法检出限为0.17~0.33μg/kg,方法定量限为0.5~1.0μg/kg,在2.0、10.0、80.0μg/kg 3个加标水平下,9种雌激素的加标回收率为65.1%~128.2%,相对标准偏差为1.9%~17.6%。该方法操作简便、快速、灵敏,重复性好,可用于大批量样品的同时快速准确检测。     

Determination of arsenic(III) and arsenic(V) by electrothermal atomic absorption spectrometry after complexation and sorption on a C-18 bonded silica column

A flow injection procedure for the separation and pre-concentration of inorganic arsenic based on the complexation with ammonium diethyl dithiophosphate (DDTP) and sorption on a C-18 bonded silica gel minicolumn is proposed. During the sample injection by a time-based fashion, the As³⁺-DDTP complex is stripped from the solution and retained in the column. Arsenic(V) and other ions that do not form complexes are discarded. After reduction to the trivalent state by using potassium iodide plus ascorbic acid, total arsenic is determined by electrothermal atomic absorption spectrometry (ETAAS). Arsenic(V) concentration can be calculated by difference. After processing 6 ml sample volume, the As³⁺-DDTP complexes were eluted directly into the autosampler cup (120 μl). Ethanol was used for column rinsing. Influence of pH, reagent concentration, pre-concentration and elution time and column size were investigated. When 30 μl of eluate plus 10 μl of 0.1% (w/v) Pd(NO₃)₂ were dispensed into the graphite tube, analytical curve in the 0.3–3 μg As l⁻¹ range was obtained (r=0.9991). The accuracy was checked for arsenic determination in a certified water, spiked tap water and synthetic mixtures of arsenite and arsenate. Good recoveries (97–108%) of spiked samples were found. Results are precise (RSD 7.5 and 6% for 0.5 and 2.5 μg l⁻¹, n=10) and in agreement with the certified value of reference material at 95% confidence level.     

Determination of triazine herbicides in surface and drinking waters by off-line combination of liquid chromatography and gas chromatography-mass spectrometry

Summary Mass spectra of 12 triazines were obtained by electron impact (EI), positive-ion chemical ionization (PCI) and negative-ion chemical ionization (NCI) using methane and isobutane as reagent gases. EI mass spectrometry is more sensitive than PCI and NCI, although the chemical ionization modes increase selectivity markedly. A pre-column packed with polymer stationary phase was employed to preconcentrate surface and drinking water samples. After desorption of the analytes with ethyl acetate, an aliquot was injected directly into the GC-MS system. Atrazine and simizine were found in these samples at 10–80 ppt levels. The limits of detection for both herbicides were below 10 ppt in drinking water.     

A flow injection-ETAAS system for the on-line determination of total and dissolved silica in waters

In this work total (Si-tot) and ‘soluble’ or reactive (Si-sol) concentrations of silicon in natural and tap waters were sequentially determined by electrothermal atomic absorption spectrometry (ETAAS). First, samples were on-line diluted based on the merging-zone principle in order to allow the determination of Si-tot within the 300–1000 μgSi l⁻¹ range. After the dilution process, a sub-sample was collected in the capillary of a sampling arm assembly (SAA). Thereafter, samples were subject to a precipitation/dissolution process in order to allow the determination of Si-sol within the 280–850 μgSi l⁻¹. Si-sol was precipitated with ammonium chloride and collected on the walls of a knotted coil. The precipitate was dissolved with ammonium molybdate in an acidic medium (HNO₃) and a sub-sample was then collected in the SAA. In both cases, 10 μl volumes of the sub-sample were injected into the atomizer with the previous introduction of 20 ng of Eu as chemical modifier (10 μl) by the spectrometer autosampler. The recovery values obtained with natural waters spiked samples were over 46% and the agreement between observed and certified samples values was good. The proportion of Si-sol in comparison with the Si-tot was high (85–95%) in most natural waters. The precision of the method was 2.4–3.5 and 4.5–6.2% (n=10) for the determination of Si-tot and Si-sol, respectively.     

Thermal lens spectrometric determination of hexavalent chromium

The applicability of thermal lens spectrometry (TLS) for quantification and routine determination of hexavalent chromium was investigated by using a collinear dual beam thermal lens spectrometer. In aqueous solutions the LOD of 0.1 μg l⁻¹ was obtained for Cr(VI) by using 160 mW laser power. The performance of the technique was verified by the determination of hexavalent chromium in standard reference water samples (NIST SRM 1643a and NIST SRM 1643c) and comparing the results for Cr(VI) in CCA (Cr, Cu, As)-treated timber extracts to concentrations obtained by atomic absorption spectrometry (AAS). Good agreement between the TLS results and reported values for Cr(VI) in SRMs as well as AAS results for Cr(VI) in CCA-treated timbers confirmed that TLS is a reliable and accurate analytical technique applicable for the determination of Cr(VI) in aqueous solutions at concentration levels 0.5–100 μg l⁻¹.     

Evaluation of ammonia as diluent for serum sample preparation and determination of selenium by graphite furnace atomic absorption spectrometry

A method for the determination of total selenium in serum samples by graphite furnace atomic absorption spectrometry was evaluated. The method involved direct introduction of 1:5 diluted serum samples (1% v/v NH₄OH+0.05% w/v Triton X-100^®) into transversely heated graphite tubes, and the use of 10 μg Pd+3 μg Mg(NO₃)₂ as chemical modifier. Optimization of the modifier mass and the atomization temperature was conducted by simultaneously varying such parameters and evaluating both the integrated absorbance and the peak height/peak area ratio. The latter allowed the selection of compromise conditions rendering good sensitivity and adequate analyte peak profiles. A characteristic mass of 49 pg and a detection limit (3s) of 6 μg 1⁻¹ Se, corresponding to 30 μg l⁻¹ Se in the serum sample, were obtained. The analyte addition technique was used for calibration. The accuracy was assessed by the determination of total selenium in Seronorm™ Trace Elements Serum Batch 116 (Nycomed Pharma AS). The method was applied for the determination of total selenium in ten serum samples taken from individuals with no known physical affection. The selenium concentration ranged between 79 and 147 μg l⁻¹, with a mean value of 114±22 μg l⁻¹.     

Speciation of halogenides and oxyhalogens by ion chromatography-inductively coupled plasma mass spectrometry

A speciation method utilizing ion chromatography coupled with inductively coupled plasma mass spectrometry is described for simultaneous analysis of eight halogenides and oxyhalogens: chloride, chlorite, chlorate, perchlorate, bromide, bromate, iodide and iodate. The method was applied for the analysis of drinking water samples collected from water treatment plants in areas in Finland, which are known to have high bromine concentrations in ground water. Water samples collected before and after disinfection were analyzed to get information about potential species conversion as a result of purification. Chloride and chlorate were the chlorine species found in these water samples, and iodine existed as both iodate and iodide. In the case of bromine, species conversion had taken place, since total bromine concentrations were increased during disinfection but bromide concentrations were decreased. No bromate was observed in the samples. The detection limits for all the chlorine species studied were 500 μg/l, for bromine species studied 10 μg/l, for iodide 0.1 μg/l and for iodate 0.2 μg/l.     

Gas chromatographic determination of nitrite in water by precolumn formation of 2-phenylphenol with flame ionization detection

Nitrite can be determined by its reaction with 2-aminobiphenyl in acidic medium to produce 2-phenylphenol which is quantified by gas chromatography with flame ionisation detection using biphenyl as an internal standard. The hydrolysis of the intermediate diazonium ion avoids many of the problems encountered in the conventional determination of nitrite by the diazotization of an aromatic amine (usually sulphanilamide) and coupling with N-(1-napthyl)ethylenediamine dihydrochloride to yield an azo dye followed by spectrophotometry. Unlike this method, the proposed reaction is rapid and does not suffer from interferences by copper(II), iron(III) and lead(II). The calibration graph was linear over the range 5–1000 μg/l NO₂-N and the limit of detection found to be 0.5 μg/l NO₂-N. A single analysis can be completed within 20 min. The method was not affected by coloured or turbid analyte solutions and has been used to determine nitrite in natural waters.     

Determination of mercury in saliva with a flow-injection system

A simple, fast and reliable method, with a low detection limit, has been developed for the determination of total mercury in saliva samples. The method uses a brominating reagent, followed by on-line addition of KMnO₄ at room temperature to convert organically bound mercury to inorganic mercury ions, and determines mercury by flow-injection cold-vapour atomic absorption spectrometry. Using the method described, complete recoveries of five mercury compounds from saliva were attained. Results obtained on real samples using the new method were comparable to that obtained using the established method with batch system. The detection limit of this method, based on three standard deviations of the blank, is 0.05 μg l⁻¹ Hg in a saliva sample of 500 μl. A sample throughput of 80 measurements per hour is possible with the method. The calibration curves are linear up to 20 μg l⁻¹ and the dynamic range extends to 40 μg l⁻¹ Hg. At a concentration of 1μg l⁻¹ mercury in saliva, the relative standard deviation is about 2% for 11 replicates; a total volume of 0.5 ml saliva is required for three replicates.     

A simple flow injection-reduced volume column system for hemoglobin typing

A flow injection (FI)-reduced volume column system was developed for hemoglobin (Hb) typing to be used as an initial screening method for thalassemia. The column was packed with 140 μl diethylaminoethyl (DEAE)-Sephadex A-50 ion exchange beads. Hb can be separated using Tris–HCl buffer solution with pH gradient 8.5–6.5 and then monitored spectrophotometrically at 415 nm. The hemolysate of 40 blood samples from packed red cells were screened for thalassemia by determining the amount of HbA₂ and HbE present. The proposed system was able to predict positive test results from those samples with β, E-trait and EE homozygous thalassemia, Hb types that were independently identified following the conventional method at the hospital laboratory. Advantages of the proposed system over the conventional column technique include low amount of reagents and blood sample needed, short analysis time and low cost. Each analysis required only 80 μl of 50 times diluted packed cells, which is equivalent to 1.6 μl undiluted packed cells, and it can be completed in only 35 min. This simple FI-reduced volume column system was demonstrated to be an economic alternative system for Hb typing to initially screen some types of thalassemia such as β-trait, E-trait and EE-homozygous which are commonly found in Thailand.     

Nitrofuran antibiotic residues in pork: The FoodBRAND retail survey

Use of nitrofuran drugs in food-producing animals has been prohibited within the EU because they may represent a public health risk. Monitoring compliance with the ban has focused on the detection of protein-bound nitrofuran metabolites which, in contrast to the parent compounds, are stable and persist in animal tissues. As part of the “FoodBRAND” project, an extensive survey of pork was undertaken across 15 European countries. Samples (n = 1500) purchased at retail outlets were analysed for the nitrofuran metabolites AOZ, AMOZ, AHD and SEM using LC–MS/MS determination of nitrobenzaldehyde derivatives. Limits of quantification for the method were 0.1 μg/kg (AOZ, AMOZ), 0.2 μg/kg (SEM) and 0.5 μg/kg (AHD). Of the 1500 samples tested, measurable residues of nitrofuran metabolites were confirmed in 12 samples (0.8% incidence overall) of which 10 samples were purchased in Portugal (AOZ, 0.3 μg/kg; AMOZ, 0.2–0.6 μg/kg) and one sample each in Italy (AMOZ, 1.0 μg/kg) and Greece (AOZ, 3.0 μg/kg).