Simple and rapid determination of phthalates using microextraction by packed sorbent and gas chromatography with mass spectrometry quantification in cold drink and cosmetic samples

A simple and rapid method using microextraction by packed sorbent coupled with gas chromatography and mass spectrometry has been developed for the analysis of five phthalates, namely, diethyl phthalate, benzyl‐n‐butyl phthalate, dicyclohexyl phthalate, di‐n‐butyl phthalate, and di‐n‐propyl phthalate, in cold drink and cosmetic samples. The various parameters that influence the microextraction by packed sorbent performance such as extraction cycle (extract–discard), type and amount of solvent, washing solvent, and pH have been studied. The optimal conditions of microextraction using C₁₈ as the packed sorbent were 15 extraction cycles with water as washing solvent and 3 × 10 μL of ethyl acetate as the eluting solvent. Chromatographic separation was also optimized for injection temperature, flow rate, ion source, interface temperature, column temperature gradient and mass spectrometry was evaluated using the scan and selected ion monitoring data acquisition mode. Satisfactory results were obtained in terms of linearity with R² >0.9992 within the established concentration range. The limit of detection was 0.003–0.015 ng/mL, and the limit of quantification was 0.009–0.049 ng/mL. The recoveries were in the range of 92.35–98.90% for cold drink, 88.23–169.20% for perfume, and 88.90–184.40% for cream. Analysis by microextraction by packed sorbent promises to be a rapid method for the determination of these phthalates in cold drink and cosmetic samples, reducing the amount of sample, solvent, time and cost.     

Determination of di(2-ethylhexyl) phthalate migration from toys into artificial sweat by gas chromatography mass spectrometry after activated carbon enrichment

The determination of di(2-ethylhexyl) phthalate migration was achieved in artificial sweat using gas chromatography mass spectrometry following activated carbon enrichment of samples. Response surface methodology (RSM) was used to optimise the conditions for maximum recovery and to understand the significance and interaction of the factors affecting the recovery of di(2-ethylhexyl) phthalate. The best compromise of analytical conditions for the simultaneous determination of analyte from spiked artificial sweat was found to be: pH (3.1), activated carbon amount (1.4 g L^?1), adsorption time (55 min) and elution solvent (chloroform). These conditions were applied to study the migration of di(2-ethylhexyl) phthalate from different children’s toys into artificial sweat. The detection limit of the method was 13.8 μg L^?1, while the relative standard deviation (%) value for the analysis of 100 μg L^?1 of the analyte was below 3.7% (n = 5).     

污水中6种邻苯二甲酸酯的测定

采用同时蒸馏萃取法提取水样中的邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄基酯、邻苯二甲酸二正辛酯、邻苯二甲酸二(2-乙基己基)酯6种邻苯二甲酸酯,用GC-MS进行定性和定量分析.对pH值、盐度和提取时间等影响因素进行优化.当pH值为2.0,NaCl加入量为5.0 g/L和提取时间为2.5 h时,目标化合物具有较好的回收率.优化条件下6种邻苯二甲酸酯的样品加标回收率为98%～105%,相对标准偏差为1.8%～5.6%,方法检出限为8.0～200.0 ng/L.方法成功应用于城市污水中该类化合物的监测.     

Simultaneous determination of 22 phthalate esters in polystyrene food‐contact materials by ultra‐performance convergence chromatography with tandem mass spectrometry

A method for the determination of 22 phthalate esters in polystyrene food‐contact materials has been established using ultraperformance convergence chromatography with tandem mass spectrometry. In this method, 22 phthalate esters were analyzed in <3.5 min on an ACQUITY Tours 1‐AA column by gradient elution. The mobile phase, the compensation solvent, the flow rate of mobile phase, column temperature, and automatic back pressure regulator pressure were optimized, respectively. There was a good linearity of 20 phthalate esters with a range of 0.05–10 mg/L, diisodecyl phthalate and diisononyl phthalate were 0.25–10 mg/L, and the correlation coefficients of all phthalates were higher than 0.99 and those of 16 phthalates were higher than 0.999. The limits of detection and the limits of quantification of 15 phthalates were 0.02 and 0.05 mg/kg, meanwhile diallyl phthalate, diisobutyl phthalate, dimethyl phthalate, di‐n‐butyl phthalate, and di(2‐ethylhexyl) phthalate were 0.05 and 0.10 mg/kg, and diisodecyl phthalate and diisononyl phthalate were 0.10 and 0.25 mg/kg. The spiked recoveries were in the range of 76.26–107.76%, and the relative standard deviations were in the range of 1.78–12.10%. Results support this method as an efficient alternative to apply for the simultaneous determination of 22 phthalate esters in common polystyrene food‐contact materials.     

Identification and quantification of trans fatty acids in bakery products by gas chromatography-mass spectrometry after dynamic ultrasound-assisted extraction

A gas chromatographic method for the identification and quantification of n-octyl esters (from n-octyl tetradecanoate to n-octyl hexa-cosanoate including dioctyl hexanedioate) and phthalates [dibutyl phthalate, benzyl butyl phthalate and di(2-ethylhexyl) phthalate] in sediments and biota from estuarine environments is described. Standards used for identification and quantification of some n-octyl esters were synthesized. The method has allowed the analysis of these compounds in polychaeta (Nereis diversicolor), oysters (Crassostea angulata), crabs (Carcinus maenas) and fish (Chelon labrosus, Platichtys flesus and Chondostroma polylepis) that were collected at different locations of the Urdaibai estuary (Bizkaia, Basque Country, Spain). Total phthalates and n-octyl esters ranged between 0.01 and 12 microg g(-1) and 0.05 and 9.4 microg g(-1), respectively, and were predominantly found in polychaeta and fish. Sediments did not contain these compounds in significant amount, only benzyl butyl phthalate, dioctyl hexanedioate and di(2-ethylhexyl) phthalate were found above limit of detection (0.01-0.05 microg g(-1)).     

Fast Chromatographic Separation of Plasticizers on Thin Layers of an Inorganic Ion-Exchanger: Quantitative Determination of Di(2-ethylhexyl)phthalate

Fast and selective separation of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, di(2-ethylhexyl)phthalate (DEHP), benzyl butyl phthalate, diisodecyl phthalate, dimethyl adipate, diethyl adipate, di(2-ethylhexyl)adipate, triethyl citrate, tributyl citrate, tributyl acetyl citrate and n-butyl stearate have been developed on thin layers of inorganic ion-exchanger stannic silicate using a mixture of toluene + ethyl acetate (10:1, v/v) as mobile phase. The development distance and time were 12 cm and 25 min, respectively. Quantitative determination of DEHP was made at wavelength 280 nm by Camag TLC Scanner-3. Limit of quantitation for DEHP was 0.50 μg per zone while its limit of detection was 0.05 μg per zone.     

Phthalates determination in pharmaceutical formulae used in parenteral nutrition by LC-ES-MS: importance in public health

A method for determining a group of phthalate esters in pharmaceutical formulae used in parenteral nutrition samples (with and without vitamins) has been developed. The phthalic acid esters (PAEs) studied were dimethyl phthalate, diethyl phthalate, butyl benzyl phthalate, dibutyl phthalate, di-(2-ethylhexyl) phthalate, and dioctyl phthalate. This group of phthalates was determined by high performance liquid chromatography (HPLC)–electrospray ionization–mass spectrometry, working in positive ion mode. The phthalates analyzed were extracted from the sample using hexane and sodium hydroxide. The hexane was then evaporated, and the compounds were redissolved in acetonitrile. The compounds were separated by HPLC working in gradient mode with acetonitrile-ultrapure water starting from 5% to 75% acetonitrile in 5 min, followed by isocratic elution for 27 min. Standard calibration curves were linear for all the analytes over the concentration range 10–250 μg L⁻¹. The method was precise (with RSD from 3.3% to 12.9%) and sensitive. The proposed analytical method has been applied to the analysis of these compounds in different pharmaceutical formulae (with different compositions) for parenteral nutrition samples in order to check the presence of phthalates and determine their concentration.     

Preparation of polypyrrole-coated magnetic particles for micro solid-phase extraction of phthalates in water by gas chromatography-mass spectrometry analysis

In this work, polypyrrole (PPy)-coated Fe(3)O(4) magnetic microsphere were successfully synthesized, and applied as a magnetic sorbent to extract and concentrate phthalates from water samples. The PPy-coated Fe(3)O(4) magnetic microspheres had the advantages of large surface area, convenient and fast separation ability. The PPy coating of magnetic microspheres contributed to preconcentration of phthalates from water sample, due to the π-π bonding between PPy coating and the analytes. Also, the coating could prevent aggregation of the microspheres, and improve their dispersibility. In this study, seven kinds of phthalates were selected as model analytes, including dimethyl phthalate (DMP), diethyl phthalate (DEP), di-iso-butyl phthalate (DIBP), di-n-butyl phthalate (DBP), benzylbutyl phthalate (BBP), di-(2-ethylhexyl) phthalate (DEHP) and di-n-octyl phthalate (DNOP), and gas chromatography-mass spectrometry (GC-MS) was introduced to detect the phthalates after sample pretreatment. Important parameters of the extraction procedure were investigated, and optimized including eluting solvent, the amount of Fe(3)O(4)@PPy particles, and extraction time. After optimization, the procedure took only 15 min to extract and concentrate analytes with high efficiency. Validation experiments showed that the optimized method had good linearity (0.985-0.998), precision (3.4-11.7%), high recovery (91.1-113.4%), and the limits of detection were from 0.006 to 0.068 μg/L. The results indicated that the novel method had advantages of convenience, good sensitivity, high efficiency, and it could also be applied successfully to analyze phthalates in real water sample.     

基质固相分散-液相色谱-质谱法测定蔬菜中的邻苯二甲酸酯

采用基质固相分散-液相色谱-质谱法测定保护地蔬菜中邻苯二甲酸酯(PAEs)含量,并分析了蔬菜中PAEs污染状况,研究了水和洗涤液浸泡对蔬菜中PAEs的消除效果。蔬菜样品经弗罗里硅土和石墨化碳黑研磨均匀后,用乙酸乙酯淋洗净化,再用液相色谱/电喷雾质谱法测定。PAEs的添加回收率为82.7%~105.7%;RSD为1.7%~6.1%;检出限为邻苯二甲酸二甲酯(DMP):0.99ng;邻苯二甲酸二乙酯(DEP):0.75ng;邻苯二甲酸二丁酯(DBP):0.70ng;邻苯二甲酸二异辛酯(DEHP):1.9ng。本方法前处理简单,具有较高的准确度和灵敏度。黄瓜、番茄、西葫芦中4种PAEs总量分别为2.02、1.26、0.91mg/kg;洗涤实验结果表明,水和洗涤液浸泡可显著降低蔬菜中的PAEs含量。     

Simultaneous screening and determination eight phthalates in plastic products for food use by sonication-assisted extraction/GC-MS methods

Studies on determination of eight kinds of phthalates, e.g. di-ethyl phthalate (DEP), di-propyl phthalate (DPP), di-isobutyl phthalate (DIBP), di-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-cyclohexyl phthalate (DCHP), di-(2-ethylhexyl) phthalate (DEHP), di-octyl phthalate (DOP), in 25 kinds of plastic products for food use, including packaging bags, packaging film, containers, boxes for microwave oven use, sucking tubes, spoons, cups, plates, etc. by gas chromatography in combination with mass spectrometry detector (GC-MS) in electronic ionisation mode (EI) with selected-ion monitoring (SIM) acquisition method (GC-MS (EI-SIM)) have been carried out. Methods have been developed for both qualitative and quantitative analysis of phthalates. Extraction, clean-up and analysis procedure have been optimized. Determination of samples were performed after frozen in liquid nitrogen and sonication-assisted extraction with hexane, clean-up with LC-C18 SPE and analyzed by GC-MS methods. The base peak (m/z = 149) of all the phthalates was selected for the screening studies. The characteristic ions, 121, 177, 222 for DEP; 191, 209 for DPP; 57, 223 for DIBP; 104 for DBP; 91, 132, 206 for BBP; 55, 167 for DCHP; 113, 167, 279 for DEHP; 279 for DOP were chosen for quantitative studies. These techniques are possible to detect phthalates at the level of 10.0 μg/kg. Overall recoveries were 82-106% with R.S.D. values at 3.8-10.2%. Only one of the 25 examined samples was free from phthalates. The rest 24 samples were found to contain at least three or more of these phthalates. The predominant phthalate detected in the studied samples was DEHP.     

Organophosphate and phthalate esters in standard reference material 2585 organic contaminants in house dust

The levels of 22 phthalate diesters (phthalates) and organophosphate triesters (organophosphates) have been investigated in standard reference material 2585 (SRM 2585) “organic contaminants in house dust.” Ultrasonic-assisted solvent extraction and solid-phase extraction on a Florisil adsorbent were used as the extraction and cleanup steps combined with analysis using gas chromatography–tandem mass spectrometry in positive ion chemical ionization mode. Seven phthalates were detected in the concentration range 1–570 μg/g. Di(2-ethylhexyl) phthalate was the major phthalate present at 570 μg/g. Ten organophosphates were detected in SRM 2585. Tris(2-butoxyethyl) phosphate was the predominant organophosphate at 82 μg/g, and nine organophosphates were determined at concentrations ranging from 0.19 to 2.3 μg/g. Five organophosphates were below the method detection limit, of which two were in level with the procedural blank. The applied extraction and cleanup method was evaluated for the analysis of SRM 2585. The extraction yield was ≥99%, except for tris(2-chloroethyl) phosphate (97%) and diethyl phthalate (98.5%). The problem of calibration curvature is addressed, and it is shown that the use of deuterated standards improves the analysis. The concentrations of ten organophosphate esters were determined in SRM 2585, and seven of these were compared with existing data. To our knowledge, this is the first report of the levels of the seven phthalates esters in SRM 2585 “organic contaminants in house dust.”     

Determination of phthalates and adipate in bottled water by headspace solid-phase microextraction and gas chromatography/mass spectrometry

The performance of three fibres for the headspace solid-phase microextraction (SPME) of di-2-ethylhexyl adipate (DEHA) and eight phthalates in water was investigated systematically under different extraction conditions. Good responses on the 65 microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fibre were observed for DEHA and all phthalates. The polydimethylsiloxane (PDMS) SPME fibre had very poor responses for the lighter and slightly polar phthalates, dimethyl phthalate (DMP) and diethyl phthalate (DEP), while the divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fibre had very poor responses for the heavier and non-polar adipate and phthalates. The salt (NaCl) was found to increase the partitioning of DMP, DEP, diisobutyl phthalate (DiBP), di-n-butyl phthalate, and benzyl butyl phthalate (BBP) from water into the headspace, while partitioning of heavier adipate and phthalates from water into headspace was suppressed when the concentration of NaCl was above 10%. The automated headspace SPME methods were developed and validated under two different salting conditions (30% NaCl for DMP, DEP and BBP, and 10% for DEHA, DiBP, DBP, di-n-hexyl phthalate (DHP), di-2-ethylhexyl phthalate (DEHP), and di-n-octyl phthalate (DOP)). Linearity with R(2) values better than 0.9949 was observed for DEHA and eight phthalates over the range from 0.1 to 20 microg L(-1). Method detection limits ranged from 0.003 microg L(-1) for DOP to 0.085 microg L(-1) for BBP. Good repeatability was observed for DEHA and most phthalates with relative standard deviation (RSD) values less than 10%. The methods were used to analyse bottled water samples for DEHA and eight phthalates. DMP, DHP, BBP, DEHA and DOP were not detected in any samples. Concentrations of the other phthalates were low (around sub-ppb) except for DBP in the water from a polycarbonate bottle at 1.72 microg L(-1).     

毛细管气相色谱法测定化妆品中的酞酸酯

建立了用毛细管气相色谱配氢火焰离子化检测器测定化妆品中6种酞酸酯(邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)、邻苯二甲酸二丁酯(DBP)、邻苯二甲酸丁基苄基酯(BBP)、邻苯二甲酸二(2-乙基己)酯(DEHP)和邻苯二甲酸二正辛酯(DOP))的方法。样品用甲醇超声提取,经高速冷冻离心,清液经干燥脱水过0.5 μm滤膜过滤,直接注入气相色谱仪进行分析。然后,经过气相色谱-电子电离-质谱检测分析,确证气相色谱-氢火焰检测的阳性检出结果。用保留时间定性,外标法定量。6种酞酸酯的回收率为82.90%～     

Blank problems in trace analysis of diethylhexyl and dibutyl phthalate: investigation of the sources, tips and tricks

The analysis of phthalates, particularly that of di(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP), is notorious for blank problems. Methods and tools are listed to identify the sources and reduce the system contamination to below 1 pg DEHP and DBP or below 1 ng mL⁻¹ of sample solution. Once direct contact with phthalate-containing plastic articles is ruled out, the air is the major source, primarily via absorption to the surfaces of laboratory glass ware. A main improvement was achieved by cleaning solvents with aluminium oxide permanently left in the reservoirs. The data enables to estimate the contamination to be expected and to design methods keeping blanks below a critical threshold.     

Validation and application of an HPLC method for determination of di (2-ethylhexyl) phthalate and mono (2-ethylhexyl) phthalate in liver samples

A reversed-phase gradient elution, UV detection method is developed for the simultaneous determination of mono (2-ethylhexyl) phthalate [MEHP] and di (2-ethylhexyl) phthalate [DEHP] in tissue samples. The method is validated with respect to extraction recovery, inter and intra-day precision, linearity of response, detect ability, and specificity. The validated method has been successfully applied to the study of DEHP and MEHP in liver, kidney, testis, brain, and plasma samples from rat.     

Determination of phthalates in beverages using multiwalled carbon nanotubes dispersive solid‐phase extraction before HPLC–MS

A microdispersive solid‐phase extraction method has been developed using multiwalled carbon nanotubes of 110–170 nm diameter and 5–9 μm length for the extraction of a group of nine phthalic acid esters (i.e., bis(2‐methoxyethyl) phthalate, bis‐2‐ethoxyethyl phthalate, dipropyl phthalate, butylbenzyl phthalate, bis‐2‐n‐butoxyethyl phthalate, bis‐isopentyl phthalate, bis‐n‐pentyl phthalate, dicyclohexyl phthalate, and di‐n‐octyl phthalate) from tap water as well as from different beverages commercialized in plastic bottles (mineral water, lemon‐ and apple‐flavored mineral water, and an isotonic drink). Determination was carried out by high‐performance liquid chromatography coupled to mass spectrometry. The extraction procedure was optimized following a step‐by‐step approach, being the optimum extraction conditions: 50 mL of each sample at pH 6.0, 80 mg of sorbent, and 25 mL of acetonitrile as elution solvent. To validate the methodology, matrix‐matched calibration and a recovery study were developed, obtaining determination coefficients >0.9906 and absolute recovery values between 70 and 117% (with relative standard deviations < 17%) in all cases. The limits of quantification of the method were between 0.173 and 1.45 μg/L. After the evaluation of the matrix effects, real samples were also analyzed, finding butylbenzyl phthalate in all samples (except in apple‐flavored mineral water), though at concentrations below its limit of quantification of the method.     

Utilization of Pine Nut Shell derived carbon as an efficient alternate for the sequestration of phthalates from aqueous system

This study highlights the importance of a cheap bio waste; Pine Nut Shell (PNS), from which a carbon is synthesized that can efficiently remove toxic phthalates from an aqueous system. PNS derived carbon shows high affinity toward phthalates in descending order along with adsorption capacity i.e., dibutyl phthalate (DBP) 5.65 mg/g > diallyl phthalate (DAP) 3.64 mg/g > diethyl phthalate (DEP) and 2.87 mg/g > dimethyl phthalate (DMP) 2.48 mg/g. Different characterization techniques such as FTIR, elemental analysis, point of zero electric charge (PZC), SEM, EDX and BET were employed to investigate the binding sites and surface area of the adsorbent. Adsorption experiments were performed both in batch and column modes. Equilibrium studies showed that the Langmuir isotherm fits best to experimental data. Kinetically, adsorption phenomena obeyed pseudo second order. Furthermore, thermodynamic results expressed the exothermic nature of adsorption on the basis of negative value of enthalpy change. Column sorption method was also adapted to check the feasibility of the adsorption process through the investigation of flow rate, breakthrough curve and pre-concentration factor which is found to be 13 for DMP and DEP and 16 for DAP and DBP. Methanol was found to be best solvent for the recovery of phthalates. Application in real water samples also showed good efficiency of PNS derived carbon for the removal of phthalates.     

New application of chitosan‐grafted polyaniline in dispersive solid‐phase extraction for the separation and determination of phthalate esters in milk using high‐performance liquid chromatography

Chitosan‐grafted polyaniline was synthesized and applied as a sorbent for the preconcentration of phthalate esters in dispersive solid‐phase extraction. By coupling dispersive solid‐phase extraction with high‐performance liquid chromatography and response surface methodology (central composite design), a reliable, sensitive, and cost‐effective method for simultaneous determination of phthalate esters including dimethyl phthalate, di‐n‐butyl phthalate, and di(2‐ethylhexyl)phthalate was developed. The morphology of sorbent had been studied by scanning electron microscopy and its chemical structure confirmed by Fourier transform infrared spectroscopy. Under optimum condition, good linearity was observed in the range of 5.0–5000.0 ng/mL. The limits of detection (S/N = 3) and limits of quantification (S/N = 10) were 0.1–0.3 and 0.3–1 ng/mL, respectively. The relative standard deviations were less than 8.8%. Finally, this procedure was employed for extraction of trace amounts of phthalic acid esters in milk samples, the relative recoveries ranged from 82 to 103%.     

Pitfalls and Solutions for the Trace Determination of Phthalates in Water Samples

A method based on liquid-liquid extraction (LLE) and automated large volume injection (LVI)-GC-MS analysis was developed for the trace determination of phthalate di-esters in water samples at sub-g L^–1 (ppb) levels. Strategies applied to reduce contamination include (i) careful selection of tools, glassware and solvents, (ii) systematic blank checks of the chromatographic system, glassware and solvents and (iii) frequent verifications of blanks during sequences. Background levels could be reduced to those present in the extraction solvent. For phthalates not present in the extraction solvent the limits of quantitation (LOQ) are 6 ng L^–1 for di-methyl phthalate (DMP), 3 ng L^–1 for benzylbutyl phthalate (BzBP) and 45 ng L^–1 for the isomeric phthalate mixtures di-isononyl phthalate (DiNP) and di-isodecyl phthalate (DiDP). For the other phthalates, the LOQ was set at twice the blank (extraction solvent) level and are 20 ng L^–1 for di-ethyl phthalate (DEP), 60 ng L^–1 for di-isobutyl phthalate (DiBP), 80 ng L^–1 for di-n-butyl phthalate (DBP) and 30 ng L^–1 for bis-(2-ethylhexyl) phthalate (DEHP).Dedicated to Professor K. Jinno on the occasion of his 60^th birthday     

Simultaneous determination of seven phthalates and four parabens in cosmetic products using HPLC-DAD and GC-MS methods

Studies on the determination of seven kinds of phthalates, i.e. diethyl phthalate, dipropyl phthalate, dibutyl phthalate, benzyl butyl phthalate, dicyclohexyl phthalate, di-(2-ethylhexyl) phthalate, and dioctyl phthalate, and four parabens, i.e. methylparaben, ethylparaben, propylparaben, and butylparaben, in 15 kinds of cosmetic products, including hair sprays, perfumes, deodorants, cream, lotion, etc., by HPLC with diode array detection and GC-MS in electron impact ionization mode with selected-ion monitoring have been carried out. Methods have been developed for both qualitative and quantitative detection of phthalates and parabens. Extraction, clean-up, and analysis procedures have been optimized. HPLC and GC-MS determinations were performed after sonication-assisted extraction with methanol and clean-up with C18 SPE. These techniques permit detection of phthalates at a level of 10.0-100.0 microg/kg and of parabens at a level of 20.0-200.0 microg/kg. Overall recoveries were 85-108% with RSD values of 4.2-8.8%. Only one of the 15 examined samples was free from phthalates and parabens. The remaining 14 samples were found to contain at least three or more of these phthalates and/or parabens. The predominant phthalates and parabens detected in the studied samples were methylparaben, propylparaben, diethyl phthalate, dibutyl phthalate, dicyclohexyl phthalate, and di-(2-ethylhexyl) phthalate. The residue level is at 1.22-5289 mg/kg.