Article pubs.acs.org/ac
Quantitative Determination of Antidepressants and Their Select Degradates by Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry in Biosolids Destined for Land Application Lydia M. Niemi, Katherine A. Stencel, Madigan J. Murphy, and Melissa M. Schultz* Department of Chemistry, 943 College Mall, The College of Wooster, Wooster, Ohio 44691, United States S Supporting Information *
ABSTRACT: Antidepressants are one of the most widely dispensed classes of pharmaceuticals in the United States. As wastewater treatment plants are a primary source of pharmaceuticals in the environment, the use of biosolids as fertilizer is a potential route for antidepressants to enter the terrestrial environment. A microsolvent extraction method, utilizing green chemistry, was developed for extraction of the target antidepressants and degradation products from biosolids, or more specifically lagoon biosolids. Liquid chromatography/tandem mass spectrometry was used for quantitative determination of antidepressants in the lagoon biosolid extracts. Recoveries from matrix spiking experiments for the individual antidepressants had an average of 96%. The limits of detection for antidepressant pharmaceuticals and degradates ranged from 0.36 to 8.0 ng/kg wet weight. The method was applied to biosolids destined for land application. A suite of antidepressants was consistently detected in the lagoon biosolid samples, and thus antidepressants are being introduced to terrestrial environments through the land application of these biosolids. Sertraline and norsertraline were the most abundant antidepressant and degradation product detected in the biosolid samples. Detected, individual antidepressant concentrations ranged from 8.5 ng/kg (norfluoxetine) to 420 ng/kg wet weight (norsertraline).
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nontarget organisms.8−10 Terrestrial organisms may also be exposed to wastewater contaminants through habitats that receive land-applied biosolids and, thus, are susceptible to nonlethal behavioral and physiological consequences, warranting further investigation.11−14 Antidepressant pharmaceuticals are one of the most heavily prescribed pharmaceutical products in the United States;15 currently more than 10% of the U.S. population uses antidepressants.16 Since antidepressants are widely prescribed in the U.S. and are incompletely removed during municipal wastewater treatment,16−21 it is not surprising that these chemicals are being detected in our waterways.17,19,21 Two studies have investigated the sorption capacities and basicity of antidepressants and discovered that antidepressants have a high likelihood to remain unchanged during wastewater treatment.22,23 Kwon and Armbrust22 investigated the sorption capacities of five selective serotonin reuptake inhibitors (SSRIs) to sediment and soil and discovered that all the studied SSRIs have high sorption capacities, except for fluvoxamine. Lajeunesse et al.23 found that most antidepressants will partition into the solid-phase sludge during wastewater treatment due to their sorption coefficients (log Kd > 4).
uman pharmaceuticals enter wastewater treatment and, subsequently, the environment, primarily by way of domestic waste from human excretion or by direct disposal of unused or expired drugs down the drain.1−3 Pharmaceutically active compounds were not commonly viewed as environmental contaminants until the late 1990s, when their active ingredients were found to negatively impact ecosystems at low nanogram per liter concentrations.4 During this time, analytical methods such as liquid chromatography/tandem mass spectrometry were being developed and optimized to measure pharmaceutically active compounds from the environment, allowing the determination of these chemicals at trace quantities. With over 3200 registered pharmaceutical ingredients, including antidepressants,5 on the market today, the occurrence of pharmaceutically active compounds in the environment has become a growing concern to the public as well as scientific and regulatory communities. Daughton and Ternes3 expressed concerns that rising pharmaceutical levels in the environment could cause irreversible change to our ecological systems. Generally, these pharmaceuticals do not display high acute toxicity because lethal effects typically occur at concentrations greater than 1 mg/L.6 However, organisms may receive continuous exposure in “pseudopersistent” scenarios where chemical half-lives are exceeded by wastewater effluent introduction rates,7 therefore justifying the need to characterize chronic, sublethal effects of these drugs on © 2013 American Chemical Society
Received: April 19, 2013 Accepted: July 11, 2013 Published: July 11, 2013 7279
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Figure 1. Chemical structures of antidepressants and degradation products, including isotopically labeled surrogate and internal standard.
concentrations of 171, 458, 62, and 42 ng/g (dry weight), respectively, in archived biosolids (2001) that were analyzed as five megacomposites representing 94 wastewater treatment plants in 32 states.27,28 Recently, Lajeunesse et al.23 measured a suite of antidepressants in treated biosolids from five different Canadian sewage treatment plants, and the detected levels were in the nanograms per gram range (dry weight); the highest mean concentration was observed for citalopram at 1033 ng/g. The aim of this study was to develop a reliable and sustainable, yet robust and quantitative, extraction method for a suite of commonly prescribed antidepressant pharmaceuticals and selected degradates in biosolids. The procedure includes microsolvent extraction, followed by liquid chromatography/ tandem mass spectrometry analysis. Extraction is often the most time-consuming step in preparation and analysis of solid and semisolid environmental samples, such as biosolids and sediments. With many extraction methods involving multiple preparatory steps, hazardous byproducts and other waste products may be produced. By elimination of unnecessary steps, waste, energy use, and resources are reduced, which translates into less solvents, chemicals, prep time, and costs.29 Microsolvent extraction was explored as a sustainable extraction technique due to its small sample volumes and solvent usage.
Also, both studies indicated that the basicity of antidepressants (pKa 9−10) increases the sorption of molecules to hydrophobic matrices.22,23 Since antidepressants have high sorption capacities,22,23 they are likely to be present in sludge and biosolids as compared to aqueous media in wastewater treatment plants. Thus, landdisposed biosolids, used for soil amendment or fertilizer, could be another source for antidepressants into (terrestrial) environments. In the United States, approximately 50% of the 6.5 million dry metric tons of sewage sludge were applied to soils in 2004.24 Of that, 75% of the total mass of land-applied biosolids was used on agricultural lands.24 Reports of the presence of antidepressants in biosolids to date is limited. Kinney et al.25 analyzed biosolids for pharmaceuticals and personal care products, including fluoxetine, the only targeted antidepressant in the study, from nine different wastewater treatment plants, and the average concentration of fluoxetine was 340 ng/g (dry weight). The U.S. Environmental Protection Agency (EPA) conducted an assessment of 145 environmental contaminants, including one antidepressant (fluoxetine), in biosolids collected nationwide between 2006 and 2007. Fluoxetine was found in 94% of the samples.26 Fluoxetine, sertraline, paroxetine, and norfluoxetine were present at mean 7280
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biosolids were taken from the lagoons in 2011, with the majority applied to land.35 Lagoon biosolid samples were collected in triplicate each month from June 2012 to February 2013. A plastic container was used to scoop the biosolids from the lagoon, and it was immediately transferred to a 1 L amber jar. The amber jars containing the lagoon biosolids were stored in a refrigerator at 4 °C until extraction, which was performed within 48 h of collection. The percentage of solids present in the collected lagoon biosolids ranged from 3.5% to 7.4%, with an average of 6.2%. Microsolvent Extraction. Within 48 h of collection, the lagoon biosolids were extracted by microsolvent extraction method in triplicate. The 1 L amber jars were inverted 10 times in order to thoroughly mix the lagoon biosolid samples. Samples (5 g) were withdrawn from the jars and added to individual 15 mL BD Falcon tubes. Fluoxetine-d5 surrogate was spiked into the sample tubes to a final concentration of 280 ng/ kg. After >2 h, 5 mL of acetonitrile containing 0.1% formic acid was added to the sample tubes. The samples were homogenized for 3 min (50% pulsing, 30% power) with a 3000 series ultrasonic homogenizer (Biologics, Inc., Manassas, VA). Then the samples were sonicated for 30 min. After sonication, the samples were centrifuged for 10 min at 10 000 rpm. The supernatant was transferred to a clean 15 mL Falcon tube and evaporated to dryness under a gentle stream of filtered air while heating at 60 °C using a N-Evap 111 (Organomation Associates, Inc., Berlin, MA). After complete sample evaporation, the samples were reconstituted to 1 mL with methanol/ water (50/50 v/v). The samples were vortexed for 2 min and sonicated again for 1 h. The 1 mL samples were transferred to amber HPLC vials and analyzed by liquid chromatography/ tandem mass spectrometry (LC/MS/MS). After LC/MS/MS analysis, samples were archived in a freezer at −20 °C. Spike and Recovery. Spike and recovery experiments were performed at two concentrations to determine the accuracy and precision of the microsolvent extraction method. The low and high sets were spiked to final concentrations of 10 and 280 ng/ kg (wet weight), respectively, of individual antidepressants and degradates. If present, the endogenous antidepressant concentrations in the lagoon biosolids were subtracted from the total quantified concentrations in the biosolids before the percent recoveries of each analyte were calculated. Liquid Chromatography/Tandem Mass Spectrometry. An Agilent 1200 series HPLC equipped with an Eclipse XDB C18 column (3.5 μm particle size, 2.1 × 150 mm) was used for separation of the lagoon biosolid samples. The autosampler injected 25 μL of sample and was set to a 30 s needle wash with acetonitrile (ACN) containing 0.1% formic acid (FA). The LC solvents were ultrapure water (solvent A) and acetonitrile (solvent B), both containing 0.1% formic acid. The LC gradient was 10 min, plus a 4 min re-equilibration time, with the flow rate set to 0.25 mL/min. The gradient was as follows: time (min) % ACN with 0.1% FA 0 0 3 0 3.1 45 4 45 6 85 7 85 7.1 95 10 95 10.1 0
The target antidepressants in this study were the commonly prescribed SSRIs and selective serotonin and norepinephrine reuptake inhibitors (SSNRIs) and included fluoxetine, sertraline, paroxetine, citalopram, venlafaxine, and duloxetine (Figure 1). The target degradates include norfluoxetine (or desmethylfluoxetine), norsertraline (or desmethylsertraline), and norvenlafaxine (or desmethylvenlafaxine). It is imperative to measure not only “parent” pharmaceuticals but also their degradation products, as they can be as pharmacologically active as the parent compound.30 For example, norvenlafaxine is not only the degradation product of venlafaxine but also the active ingredient in Pristiq, a current prescription antidepressant.31 The validated methodology was then applied to biosolids, or more specifically lagoon biosolid samples, destined for land application. Long-term effects of the land application of biosolids on the environment have not been studied sufficiently; therefore investigation into this area is necessary to fully understand the potential risks.11,22,25,27,32,33
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EXPERIMENTAL SECTION Standards and Reagents. All native and isotopically labeled antidepressant solid standards (>98% purity) were purchased from Toronto Research Chemical Inc. (North Fork, ON, Canada). HPLC-grade methanol and acetonitrile were obtained from Pharmoco-Aaper (Brookfield, CT). Formic acid (88%) was obtained from Fisher Scientific (Fairlawn, NJ). A Nanopure Diamond water purification system (VWR International, West Chester, PA) dispensed ultrapure water. Individual stock solutions of the native and isotopically labeled antidepressant compounds were prepared in MeOH. All stock solutions were stored in amber bottles in a freezer at −20 °C. Glassware Cleaning. All glassware was cleaned according to guidelines from the U.S. Geological Survey.34 Briefly, the glassware was soaked (>1 h) in 1 g of Alconox/L of hot tap water. Tap water was used to rinse the glassware until no soap residue was visible. The glassware was then rinsed three times with ultrapure water and three times with the solvent of use. Environmental Sample Collection. Biosolids samples, or more specifically lagoon biosolid samples, were collected at the Water Pollution Control Plant (WPCP) in Wooster, OH. This plant serves a community of approximately 26 000 people and treats an average wastewater flow of 17 million L/day.35 The plant uses activated aerobic sludge as the secondary treatment and anaerobic digestion. UV disinfection has recently been installed for tertiary treatment. In 2011 the plant removed 2.7 million kg of suspended solids from wastewater.35 After undergoing treatment in the activated aerobic sludge (>20 h) and anaerobic digestion for approximately 20 days, the biosolids are then added to the lagoon. The lagoon is a storage basin for the digested biosolids until land application can occur, which is dependent upon weather, crop rotation, and harvesting cycles. No additional treatment process that is controlled by the plant occurs in the lagoon; however, additional treatment may occur through photolysis and/or by microbial degradation by bacteria present in the lagoon. The WPCP adds digested biosolids to the lagoon almost every day, about 190 000 L, and decants excess water back to the headworks. A contract hauler mixes the lagoon biosolids with residual lime and soda ash to ensure that the total solids are at least 6% before land application.35 The biosolids are directly taken from the lagoon throughout the year and are applied to local agricultural fields and in two nearby counties.35 More than 53 million L of 7281
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extrapolation of the trendline equation of each analytes’ respective calibration curve. The noise concentrations were multiplied by 3 to determine the LOD and by 10 for the LOQ. Any analyte detected above the LOD but below the LOQ was reported as ≤LOQ.
The Agilent triple quadrupole 6410 mass spectrometer was operated in positive electrospray ionization (ESI) mode with the gas temperature and flow set to 350 °C and 8 L/min, respectively. The nebulizer pressure was operated at 35 psi, the spray needle was held at 0 V, and the spray chamber was held at −4500 V. The mass spectrometer was operated in multiplereaction-monitoring (MRM) mode, which scanned for both primary and secondary product ions of the precursor ion of each target analyte (Table 1).
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RESULTS AND DISCUSSION Biosolid Sample Preparation Optimization. Initial sample preparation of the lagoon biosolid samples for this study utilized the method of accelerated solvent extraction (also referred to as pressurized liquid extraction). The antidepressants were extracted by accelerated solvent extraction using a method described elsewhere for pharmaceuticals and personal care products, which included the antidepressant fluoxetine,25 and is briefly described in the Supporting Information. Due to the poor extraction recoveries and the large amount of solvent accrued, other extraction techniques were explored. Microscale solvent extraction was a desirable alternative to accelerated solvent extraction for this study. It is also a green technique as it employs small sample volumes and solvent usage, thereby reducing generated waste and time-consuming preparatory steps. Microscale solvent extraction has been used for a wide variety of target chemicals; a brief literature search reports that microscale solvent extraction has been used for pesticides,36 perchlorate,37 and lipids.38 Since microscale solvent extraction has few preparatory steps, there are few parameters to optimize. For this study, 1 and 5 g sample sizes were compared. The target analytes were sufficiently detected in the lagoon biosolids at both volumes; however, the 5 g sample volume was chosen as it provided a larger signal-to-noise ratio for the antidepressants as compared to the 1 g sample volume. The solvents methanol and acetonitrile both produced similar extraction efficiencies; however, acetonitrile was chosen as the extraction solvent as it had better reproducibility. Acidification was previously demonstrated to to increase recovery efficiency in antidepressants;19 therefore, 0.1% formic acid was added to the acetonitrile. The accuracy and precision of the extraction method was determined from spike and recovery experiments performed at high (280 ng/kg) and low (10 ng/kg) concentrations in the WPCP lagoon biosolids. Recoveries for all analytes typically were good with an average of 96% for the high and low sets; however, some notable outliers were observed, leading to a range of 12−160% (Table 2). The lower observed recoveries of sertraline and norsertraline may be explained by their high sorption capacities to sludge.23 The recoveries from this microsolvent extraction method are comparable to previously reported values in the literature for methods that include more involved sample preparations of antidepressants from biosolids. For example, fluoxetine and sertraline have 100% and 74% recoveries, respectively, with EPA Method 1694, which is sequential liquid−liquid extractions followed by solid-phase extraction.26,28 Also, Lajeunesse et al.23 reported recoveries of 44−101% for antidepressants and degradates using minimal sample preparation followed by solid-phase extraction, and Radjenović et al.39 reported recoveries of 16% and 59% for fluoxetine and paroxetine, respectively, in sludge samples using accelerated solvent extraction followed by solid-phase extraction. The limit of detection (LOD) and limit of quantitation (LOQ) of the microsolvent extraction method were determined for each antidepressant and degradation product
Table 1. Mass Spectrometer Parameters and Ion Transitions Used for Identification and Quantitation of Antidepressants and Their Degradation Productsa primary
a
secondary
compd
precursor ion, m/z
product ion, m/z
collision energy, eV
product ion, m/z
collision energy, eV
fluoxetine-d5b fluoxetine sertraline venlafaxine citalopram paroxetine duloxetine norfluoxetine norsertraline norvenlafaxine
315 310 306 278 325 330 298 296 292 264
44 44 159 260 262 192 154 30 275 246
15 10 15 10 15 15 15 15 15 15
148 148 275 121 109 70 44 134 159 58
15 15 10 10 15 15 15 15 15 15
Fragmentor voltage was 60 V for all compounds. bSurrogate.
Quantitation and Confirmation. After LC/MS/MS analysis, all data were processed with MassHunter Quantitative software. Calibration curves with concentrations ranging from 1 to 1000 ng/kg were used for quantification of the target antidepressants and fluoxetine-d5 in the samples. The quantitative software used the weighted (1/x) linear leastsquares regression model to generate trend lines. This model minimizes the influence of lower concentration calibration standards in order to decrease the effect of measurement uncertainties on the correlation coefficient. All calibration curves contained a minimum of six data points and were not forced through 0. All correlation coefficients were high (R2 ≥ 0.99). The calibration curves were analyzed with each sample batch run on the LC/MS/MS. All concentrations of antidepressants in the lagoon biosolids were reported as wet weights. The reason for choosing wet weight over the more commonly used dry weight is 2-fold. First, the biosolids were extracted as collected, which were “wet” samples, approximately 6% solids. To report a true dry weight of the analyte in the biosolids, the sample must be dried prior to sample preparation. Second, the contract hauler does not further dry the lagoon biosolids before their land application. Therefore, by measuring the antidepressants and degradates in the biosolids as they are applied (∼6% solids), a better representation is provided of their occurrence and levels being introduced to terrestrial environments in both the adsorbed and dissolved phases of biosolids. To determine the limit of detection (LOD) and limit of quantitation (LOQ) of each antidepressant and degradation product, the average noise level for each analyte was determined from the chromatograms within the biosolid matrices. The average noise signals were converted to concentration values (nanograms per kilogram) through 7282
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have the highest sorption coefficient (log Kd = 4.5) as compared to the other SSRIs and SSNRIs. Venlafaxine and duloxetine were not always detected above the LOQ. Fluvoxamine was never detected in the lagoon biosolid samples and thus was not included in the method validation. This could be due to the values of sorption capacities for these antidepressants. Kwon and Armbrust22 found that fluvoxamine had the lowest sorption capacity of the SSRI antidepressants. Lajeunesse et al.23 and Hörsing et al.40 determined that the sorption coefficient for venlafaxine was 2 orders of magnitude smaller than that for sertraline. An alternative explanation for the low observed levels could be that these antidepressants are more susceptible to degradation. In the case of venlafaxine, its degradation product norvenlafaxine was observed and measured in every monthly sample. With the exception of the September sample, norvenlafaxine was detected at higher concentrations than venlafaxine (Table 3 and Figure 3). The higher observed concentrations for norvenlafaxine as compared to venlafaxine could be explained by its use as the active ingredient in the current antidepressant Pristiq.31 Alternatively, the higher abundance of norvenlafaxine could be explained by the degradation of venlafaxine. In microcosm studies conducted with aerobic wastewater sludge collected from the WPCP plant, venlafaxine was shown to significantly degrade after 2 days of treatment (unpublished data). The variable concentrations and large standard deviations of the antidepressants detected in lagoon biosolid samples collected monthly between June 2012 and February 2013 were expected due to the heterogeneous nature of the biosolids (Table 3 and Figure 3). There is not a standard residence time for the biosolids in the lagoons. Until the lagoon biosolids are taken for land application, they remain in the lagoon and mix with the constantly inflowing biosolids, which enter primarily from the anaerobic digestors.35 Other contributing factors to the heterogeneous character of the lagoon biosolids include the plant residual lime and soda ash, which is added at nonuniform rates and locations, and the fact that lagoons are open air and susceptible to dilution from precipitation events. These factors impact the concentrations of the individual antidepressants detected in the monthly lagoon biosolid samples. A suite of antidepressants was consistently detected in the lagoon biosolid samples, and thus, antidepressants are being introduced to terrestrial environments through the land application of these biosolids. Temperature has also been found to impact the survival of antidepressants during wastewater treatment. Lajeunesse et al.23 discovered that the efficiency of antidepressant degradation by microorganisms decreased at low temperatures. In a wastewater treatment plant in Canada, bioremediation was found to be 11% less efficient for removal of antidepressants at 11.3 °C (April) than at 21.2 °C (September).23 Seasonal variation between sampling therefore could impact the concentrations of antidepressants in the lagoon biosolids, where high temperatures correspond to reduced antidepressant concentrations. However, this trend was not observed in this study, where the highest concentrations of antidepressants in the biosolids were from samples collected in December, one of the colder months, when the average high temperature in northeast Ohio is 3 °C (Table 3 and Figure 3). Overall, there were no observed seasonal trends for the occurrence of antidepressants in biosolids.
Table 2. Mean Percent Recoveries Obtained after Extraction, and Limits of Quantitation and Detection, of Individual Antidepressants and Degradates in Biosolids mean recovery (% ± SD) compd fluoxetine-d5b fluoxetine sertraline venlafaxine citalopram paroxetine duloxetine norfluoxetine norsertraline norvenlafaxine
low spikea
high spikea
nac 74.8 58 88.7 30.7 127.7 106.4 120 12 137
70 102 62 108 105 142.5 130 118 74 160
± ± ± ± ± ± ± ± ±
0.9 6 0.3 0.8 0.4 0.5 1 1 3
± ± ± ± ± ± ± ± ± ±
10 4 6 6 5 0.4 1 2 1 20
LOQ (ng/ kg)
LOD (ng/ kg)
na 18 15 9.1 1.2 7.0 6.3 6.0 27 11
na 0.66 3.0 2.7 0.36 2.1 1.9 1.8 8.0 3.3
Low and high sets were spiked to final concentrations of 10 and 280 ng/kg (wet weight), respectively, of individual antidepressants and degradates. bSurrogate. cna = not analyzed.
a
in lagoon biosolids. LOD and LOQ were defined as the concentration of each antidepressant analyte that produced a signal-to-noise ratio of 3:1 and 10:1, respectively, in the lagoon biosolid samples. The LOD values were determined to range from 0.36 (citalopram) to 8.0 (norsertraline) ng/kg wet weight (Table 2). The individual LOQs ranged from 1.2 (citalopram) to 27 (norsertraline) ng/kg wet weight. Liquid Chromatgraphy/Tandem Mass Spectrometry. Chromatographic separation of the detected target antidepressants and degradation products was achieved for the calibration standards (not shown) and for the lagoon biosolid samples (Figure 2). The ratio of the two product ions (quantitation and confirmation ions) from fragmentation of the target analyte (precursor ion) in combination with the retention time was used to identify the target antidepressants. The quantitation ion was defined as the most abundant product ion for each of the analytes, and its concentration was reported for the target antidepressants (Table 3). Application to Environmental Samples. The range of concentrations of detected target antidepressants varied from 8.5 ± 0.7 (norfluoxetine) to 420 ± 100 (sertraline) ng/kg wet weight in the lagoon biosolid samples (Table 3). Of the six antidepressants and three degradates that were detected, sertraline and its primary degradation product, norsertraline, were the most abundant analytes measured. Fluoxetine, citalopram, paroxetine, norfluoxetine, and norvenlafaxine were also detected every month in addition to sertraline and norsertraline. A comparison of the antidepressants present in the WPCP lagoon biosolids to those previously reported in biosolids show similar antidepressant profiles. Sertraline and norsertraline were the first and second most abundant antidepressants observed in a recent study that looked for six antidepressants in biosolids collected from anaerobic digestion tanks from a wastewater treatment plant in Ithaca, NY.33 The detected concentrations for sertraline and norsertraline were 104 and 93 ng/g (dry weight), respectively.33 Sertraline was also the most abundant antidepressant measured in the archived composite biosolids from the 2001 EPA National Sewage Sludge Survey.28 The prevalence of sertraline and its primary degradation product in sludge may be explained, in part, by their high sorption capacities to wastewater solids.22,23 In the study of Lajeunesse et al.,23 sertraline was determined to 7283
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Figure 2. Representative LC/MS/MS chromatogram of antidepressants and degradates determined in lagoon biosolids (August 2012 sample). For each target antidepressant and degradate, two precursor-to-product ion transitions were monitored, a quantitative and a qualitative transition, to increase specificity. Both monitored ion transitions are shown in the representative chromatogram for each antidepressant or degradate. A box is used to identify peaks that correspond to the target analyte, according to retention times determined by reference standards.
Table 3. Mean Concentrations for Antidepressants and Degradation Products in Monthly Lagoon Biosolid Samples Collected from June 2012 to February 2013 mean concn (n = 3), ng/kg wet weight ± SD sertraline fluoxetine venlafaxine citalopram paroxetine duloxetine norsertraline norfluoxetine norvenlafaxine
overall mean
June
July
Aug
Sept
Oct
Nov
Dec
Jan
Feb
230 ± 40 60 ± 10 75 ± 40 110 ± 20 34 ± 2 25 ± 4 200 ± 30 45 ± 3 70 ± 10
120 ± 13 70 ± 50
