A novel metallobridged bis(b-cyclodextrin)s fluorescent probe for the determination of glutathione Bo Tang, Fang Liu, Kehua Xu and Lili Tong College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, China Reduced glutathione (GSH: c-Glu-Cys-Gly), a princi- pal non-protein thiol compound, plays an important role in many biological processes such as transport, protein synthesis, catabolism and metabolism [1]. It can also protect cells against reactive oxygen species and help them maintain an adequate intracellular redox status [2]. Thus, the quantitative detection of GSH is very important for investigating biological pro- cesses. Many methods have been developed to deter- mine GSH, including HPLC [3,4], electrochemistry [5,6], spectrofluorimetry [7,8] and so on. Although these methods are currently available for GSH deter- mination, most of them are complicated and inconve- nient to operate. The determination of GSH in plasma is particularly challenging because redox conditions change rapidly after blood collection [9,10]. Therefore, a rapid and simple method for the analysis of GSH in plasma is needed. Cyclodextrins (CDs) are a class of cyclic oligosac- charides with six to eight d-glucose units linked by a-1,4-glucose bonds. They possess a hydrophobic cavity capable of including a variety of hydrophobic compounds via host–guest complexation [11]. They are also widely used as a solubilizer because of their hydrophilic exterior [12,13]. Among various functional CDs, bridged bisCDs, which comprise two CD cavities linked by a functional bridged has received great attention [14]. In comparison with native CDs and mono-modified CDs, bridged bisCDs exhibit signifi- cant high-binding ability and molecular recognition through the cooperative binding of two adjacent CD units [15]. Furthermore, metallobridged bis(b-CD)s can afford more stable inclusion complexes with guest molecules through the cooperative binding of two b-CD cavities and the additional interactions between the coordinated metal and the guest molecule [16]. Keywords competitive complexation; glutathione; metallobridged bis(b-cyclodextrin)s; molecular recognition; spectrofluorimetry Correspondence B. Tang, College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, China Fax: +86 531 8618 0017 Tel: +86 531 8618 0010 E-mail: tangb@sdnu.edu.cn (Received 30 November 2007, revised 13 January 2008, accepted 25 January 2008) doi:10.1111/j.1742-4658.2008.06310.x A novel metallobridged bis( b-cyclodextrin)s 2 [bis(b-CD)s 2] was synthesized and characterized by means of 1 H NMR, IR, element analysis and redox iodometric titration. The fluorescence of metallobridged bis(b-CD)s 2 was weak compared with bis(b-CD)s 1 because of the paramagnetism of copper (II) ions. Glutathione was able to form complexes with copper (II) derived from the metallobridged bis(b-CD)s 2. This competitive complexa- tion with copper (II) may lead to a significant fluorescence recovery of the bis(b-CD)s. Therefore, a rapid and simple spectrofluorimetric method was developed for the determination of glutathione. The analytical application for glutathione was investigated in NaCl ⁄ P i (pH 6.00) at room temperature. The linear range of the method was 0.30–20.0 lmolÆL )1 with a detection limit of 63.8 nmolÆL )1 . There was no interference from the plasma constituents. The proposed method had been successfully used to determine glutathione in human plasma. Abbreviations bis(b-CD), bis(b-cyclodextrin); CD, cyclodextrin; GSH, glutathione. 1510 FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS In this study, we synthesized a novel fluorescent bis(b-CD)s 1 containing two metal-binding sites and two naphthyl fluorophores (Scheme 1). The compound showed satisfactory water solubility because of the two b-CDs. Complexes 2 were formed when copper (II) ions were added to bis(b-CD)s 1, at the same time, flu- orescence quenching was discovered. Afterwards, the addition of GSH to 2 induced a recovery of fluores- cence (Scheme 2). Based on this principle, we devel- oped a rapid and simple spectrofluorimetric method for the analysis of GSH. The proposed method has been successfully applied to the determination of GSH in human plasma. Results and Discussion Metal coordination and stoichiometry Job’s experiments were performed to explore the coor- dination stoichiometry of the bis(b-CD)s 1–copper (II) complex in aqueous solution as described previously [16]. A representative Job’s plot for the coordination of bis(b-CD)s 1 with copper (II) chlorate is shown in Fig. 1. The plot for the 1 ⁄ Cu 2+ system showed a maximum at 0.67 which corresponded to a 1 ⁄ Cu 2+ stoichiometry of 1 : 2. This indicates that one Scheme 1. Synthesis of the novel metallo- bridged bis(b-CD)s. Scheme 2. The detection mechanism. 0.2 0.4 0.6 0.8 1.0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 Relative absorbance (a.u.) [Cu 2+ ] / ([Cu 2+ ]+[1]) Fig. 1. Job’s plot of the 1 ⁄ Cu 2+ system at 349 nm. [1] + [Cu 2+ ] = 1.00 · 10 )4 molÆL )1 ; pH 6.00. B. Tang et al. Glutathione determination using bis(b-CD)s FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS 1511 bis(b-CD)s 1 could bind two copper (II) ions, as illus- trated in Scheme 1. Excitation and emission spectra Following the procedure described below, the excita- tion (left) and emission (right) spectra were scanned (Fig. 2). The maximum excitation and emission wave- lengths were 365 and 480 nm respectively. The fluores- cence intensity of metallobridged bis(b-CD)s 2 was weak compared with bis(b-CD)s 1. A significant recov- ery of fluorescence was observed when GSH was added to metallobridged bis(b-CD)s 2 in this analytical system. Influence of pH Because of the instability of CD and the amido bond at very low pH, the use of strongly acidic solution was avoided [17]. Moreover, copper (II) will deposit in alkali solution. Thus the optimal pH of the system was in the range 4.00–9.00. The results are shown in Fig. 3. As can be seen, the fluorescence intensity was relatively high and remained almost constant over the pH range 5.00–6.50. Therefore, a pH of 6.00 was fixed using NaCl ⁄ P i buffer. The effect of the buffer is lost if too small a quantity is used. Whereas if the amount of buffer is excessive, the ionic strength is too great, which influences the flu- orescence intensity. Therefore, the influence of the vol- ume of buffer was measured. Because the volume of buffer added (1.00–3.00 mL) had little effect on the fluorescence intensity, 2.00 mL of buffer was chosen in subsequent experiments. Influence of the concentration of metallobridged bis(b-CD)s 2 The influence of the concentration of 2 on fluorescence intensity is shown in Fig. 4. As can be seen, as the concentration of 2 increased, the fluorescence intensity of the system also increased slightly. We therefore used 2.00 mL of 2.00 · 10 )4 molÆL )1 metallobridged bis(b- CD)s 2. Influence of reaction time The effect of reaction time was studied, the result (Fig. 5) showed that the fluorescence intensity reached a maximum after the reagents had been added for 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 0 500 1000 1500 2000 2500 3000 3500 4000 4500 3 3 2 2 1 1 1:bis(β-CD)s1 2:metallobridged bis( β-CD)s2 3:metallobridged bis( β-CD)s2+GSH Fluorescence intensity (a.u.) Wavelength (nm) Fig. 2. Excitation (left) and emission (right) spectra. [bis(b- CD) 1] = 2.00 · 10 )5 molÆL )1 ; [metallobridged bis(b-CD) 2]= 2.00 · 10 )5 molÆL )1 ; [GSH] = 5.00 · 10 )6 molÆL )1 ; pH 6.00. 150 200 250 300 350 Relative fluorescence intensity (a.u.) 4.00 5.00 6.00 7.00 8.00 9.00 pH Fig. 3. Influence of pH on the fluorescence intensity. [GSH] = 2.00 · 10 )6 molÆL )1 ;[2] = 4.00 · 10 )5 molÆL )1 . 160 180 200 220 240 260 280 300 320 340 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Relative fluorescence intensity (a.u.) [metallobridged bis(β-CD)s 2] (10 –5 mol·L –1 ) Fig. 4. Influence of the concentration of 2 on fluorescence inten- sity. [GSH] = 2.00 · 10 )6 molÆL )1 ; pH 6.00. Glutathione determination using bis(b-CD)s B. Tang et al. 1512 FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS $ 10 min and remained constant for at least 1 h. Hence, the reaction was left to proceed for 10 min, and the fluorescence was then measured at room tem- perature. Influence of interference The influence of the main constituents of plasma on the determination of 2.00 · 10 )6 molÆL )1 GSH were studied. The criterion for interference was fixed at a ± 5.0% variation in the average fluorescence intensity calculated for the established level of GSH. A 3000- fold mass excess of plama over 2.00 · 10 )6 molÆL )1 GSH was tested first. If interference occurred, the ratio was gradually reduced until interference ceased. The results are shown in Table 1 and it can be seen that the determination was free from interference by the constituents of plasma. Mechanism The novel fluorescent bis(b-CD)s 1 contained two strong coordination sites for copper (II) ions and two naphthyl fluorophores. The compound could be dis- solved in aqueous solution and showed high binding ability because of the two adjacent bis(b-CD)s. Because of the conformation of the linker of 1, the nitrogen atoms and amido bond formed two chelate rings to coordinate with copper (II) ions. This coordi- nation effect and the paramagnetism of copper (II) ions induced fluorescence quenching. However, GSH has a great propensity for forming complexes with metal ions that have strong electrophilic characteristics [18], such as copper (II) [19,20], mercury [21] and cadmium [22]. In this system, the quenched copper (II) complex 2 could interact with the thiol and amino of GSH via a cooperative chelation effect [23,24], which led to recovery of the fluorescence intensity of bis(b- CD)s. Based on this principle, we developed a spectro- fluorimetric method with high selectivity to determine GSH in human plasma. Analytical characteristics Under optimum experimental conditions, there was a linear relationship between fluorescence intensity and GSH concentration in the range 0.30–20.0 lm with a correlation coefficient of 0.9976 (Fig. 6). The regres- sion equation was F = 2644.17 + 63.98 [GSH] (lm). The detection limit, as defined by IUPAC [25], was determined to be 63.8 nmolÆL )1 , according to the formula C = KS 0 ⁄ S, where K = 3 (standard devia- tion = 1.36), obtained from a series of 11 reagent blanks, and S is the slope of the standard curve. The relative standard deviation was 2.5%, obtained from a series of 11 standards each containing 2.00 lm GSH. When the concentration of GSH exceeded that of metallobridged bis(b-CD)s 2 by as much as 100-fold, a decrease in fluorescence intensity was discovered. This is consistent with that previously reported by Liu et al. [16]. 10 20 30 40 50 60 100 120 140 160 180 200 220 240 260 280 300 320 340 0 Relative fluorescence intensity (a.u.) Time (min) Fig. 5. Effect of reaction time on fluorescence intensity. [GSH] = 2.00 · 10 )6 molÆL )1 ;[2] = 4.00 · 10 )5 molÆL )1 ; pH 6.00. Table 1. Interferences of various coexisting biological substances. Coexisting substance Concentration (molÆL )1 ) Relative error (%) K + 6.0 · 10 )3 )3.7 Na + 6.0 · 10 )3 )3.0 Ca 2+ 1.6 · 10 )3 1.4 Mg 2+ 2.4 · 10 )4 4.2 Zn 2+ 6.0 · 10 )4 1.2 Fe 2+ 1.0 · 10 )4 )4.4 Fe 3+ 6.0 · 10 )5 )3.6 Arginine 1.0 · 10 )4 3.5 L-Phenylalanine 2.0 · 10 )5 4.6 Lysine 2.0 · 10 )5 3.1 L-Cysteine 1.0 · 10 )4 4.3 Tyrosine 1.0 · 10 )4 )4.5 Bilirubin 2.0 · 10 )5 4.8 Glucose 6.0 · 10 )5 )2.6 Cholesterol 1.0 · 10 )4 3.5 Ascorbic acid 1.0 · 10 )3 )3.3 Uric acid 4.0 · 10 )4 4.1 Gly–Leu 2.0 · 10 )4 2.7 Gly–Gly–Gly 2.0 · 10 )4 1.5 Gly–Gly 2.0 · 10 )4 2.3 Gly–Phe 8.0 · 10 )5 4.4 Gly–Pro 6.0 · 10 )4 3.5 Leu–Gly–Gly 8.0 · 10 )4 2.9 Leu–Gly 8.0 · 10 )4 4.3 B. Tang et al. Glutathione determination using bis(b-CD)s FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS 1513 Applications Sample collecting and processing Fasting venous blood (5.00 mL) was routinely col- lected from the author Y. Liu, and transferred to a 10 mL centrifuge tube containing heparin sodium as an anticoagulant. The blood was immediately centri- fuged at 1000 g for 1 min at room temperature to remove cells and platelets [10]. Afterwards, 0.50 mL of absolute alcohol was added to the plasma with shaking. Plasma proteins were precipitated and removed by centrifugation. The final plasma samples used in the determination of GSH were obtained. Determination of GSH in plasma and accuracy assessment by recovery experiments In order to evaluate the applicability of the proposed method, fluorescence determination in plasma was per- formed according to the following procedures. Into a series of 1.00-mL Eppendorf microtubes were sequen- tially added different aliquots of the plasma samples, GSH stock solution (3.00 · 10 )4 molÆL )1 ), 0.04 mL of 1.00 · 10 )3 molÆL )1 metallobridged bis(b-CD) 2 and 0.20 mL of 0.10 molÆL )1 NaCl ⁄ P i (pH 6.00). The experimental data are shown in Table 2. The mixture was diluted to mark with ultra-pure water, shaken thoroughly and equilibrated at room temperature for 10 min. The fluorescence intensity of the solution was measured at 365 ⁄ 480 nm. The GSH content of the plasma was derived from the standard curve and the regression equation. The average recovery test was made using the standard addition method, and the RSD was generally good when obtained from a series of six plasma samples. These results are also given in Table 2. Compared with previously reported methods (Table 3), our results indicate that the recovery and precision of our method of determining GSH in plasma are satisfactory. Conclusions We synthesized a novel metallobridged bis(b-CD)s 2, which afforded two hydrophobic binding sites coopera- tively associating with the guest GSH and also provided additional binding interactions between the hetero- atoms of GSH and the coordinated metal center. GSH was able to form complexes with copper (II) derived from the metallobridged bis(b-CD)s 2. This competitive 0246810121416182022 2600 2800 3000 3200 3400 3600 3800 4000 Fluorescence intensity (a.u.) [GSH] (µM) Fig. 6. Linear plot of fluorescence intensity with increase in GSH concentration. [2] = 4.00 · 10 )5 molÆL )1 ; pH 6.00. All spectra were obtained under the optimum experimental conditions at 365 ⁄ 480 nm and room temperature. Table 2. GSH determination in plasma samples (n =6,P = 95%). Samples Plasma (mL) GSH added (l M) Measured a (lM) RSD (%) Recovery (%) GSH content of plasma (lM) A 0.25 0 0.94 4.43 ) 3.77 B 0.50 0 2.02 3.97 – 4.05 C 0.75 0 3.66 3.56 – 4.88 A¢ 0.25 9.00 10.3 2.58 104 3.77 B¢ 0.50 6.00 7.94 3.47 98.7 4.05 C¢ 0.75 3.00 6.80 1.62 105 4.88 a Mean of six determinations using the proposed method. Table 3. Analytical characteristics compared with other methods reported. Methods Linear range (l M) Limit of detection GSH in plasma (lM) HPLC [26] 0.81–13.02 0.13 l M 3.39 ± 1.04 HPLC with fluorimetry [27] 0.2–20.0 14 f M 1.82 ± 0.55 Proposed method 0.30–20.0 63.8 n M 4.01 ± 0.38 Glutathione determination using bis(b-CD)s B. Tang et al. 1514 FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS complexation with copper (II) may lead to a fluores- cence recovery of the bis(b-CD)s. Based on this princi- ple, we developed a spectrofluorimetric method with high selectivity to determine GSH. The linear range of the method was 0.30–20.0 lmolÆL )1 with a detection limit of 63.8 nmolÆL )1 . There was no interference from the plasma constituents. The proposed method was suc- cessfully used to determine GSH in human plasma. Experimental procedures Apparatus and reagents All spectrofluorimetric measurements were carried out with an Edinburgh FLS920 spectrofluorimeter (Edinburgh Instru- ments Ltd, Livingston, UK) equipped with a xenon lamp and 1.0 cm quartz cell. Absorption spectra were obtained from UV-1700 (Shimadzu, Kyoto, Japan) UV–visible spectroph- tometer. Infrared spectra were obtained from a PE-983G IR- spectrophotometer (Perkin-Elmer, Palm Springs, CA, USA). 1 H NMR spectra were recorded on a Bruker Avance 300, elemental analysis was performed on Perkin-Elmer Series P CHNS ⁄ O analyzer. pH measurements was made with a pHS- 3 digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass-calomel electrode. Centrifuga- tion was carried out on a of Sigma 3K 15 centrifuge. Reduced glutathione (99.8%) (Sigma, Mannheim, Ger- many) was used without further purification. A stock solution (1.00 · 10 )3 molÆL )1 ) of GSH was prepared with ultra-pure water. b-CD (China Medicine Group Shanghai Chemical Reagent Corporation, Shanghai, China) was puri- fied by recrystallizing twice in ultra-pure water, followed by vacuum drying at 95 °C for 24 h. 3-Amino-2-naphthoic acid (Alfa Aesar, Word Hill, MA, USA) was used without further purification. Mono(6-p-toluenesulfonyl- 6-deoxy)-b-cyclodextrin was prepared by reacting p-tosyl chloride with b-CD in dry pyridine as described previously [28,29]. Mono(6-p-toluenesulfonyl-6-deoxy)-b-cyclodextrin was then converted to mono(6-aminoethylamino-6-deoxy)- b-CD with 57.1% yield upon heating in excess ethylenedi- amine at 75 °C under nitrogen for 7 h [30]. Compound 3, oxamide bis(2-naphthyl) acid, was prepared according to the procedure reported previously [31]. Other chemicals used were of analytical reagent grade. The water used in this study was purified using a Mill-Q (18.2 MWÆcm )1 ) water system. A 100 k Nanosep filter (Pall Corp., East Hills, NY, USA) and micoron YM—30-30000 NMWL (Millipore, Billerica, MA, USA) were used as ultra-purification instrumentation. Synthesis of the novel bis(b-CD)s Synthesis of compound 1 Mono (6-aminoethylamino-6-deoxy)-b-CD (2.00 g) was dis- solved in dimethylformamide (50 mL) in the presence of a small amount of 0.4 nm molecular sieves, and then 3 (0.21 g) was added. The mixture was stirred for 24 h at 70 °C under nitrogen. It was then allowed to stand for 5 h until no further precipitate was deposited. The precipitate was removed by filtration, and the filtrate evaporated to dryness under reduced pressure. The residue was dissolved in a minimum amount of hot water and poured into ace- tone to give an orange precipitate. The orange precipitate was purified by three recrystallization steps in ultra-pure water. After the residue had been dried under a vacuum, pure sample 1 was obtained with a 27% yield. UV ⁄ vis k max (H 2 O) ⁄ nm (log e): 348 (1.62). 1 H NMR (300 MHz, DMSO- d 6 , TMS ppm): d2.00–3.00 (m, 14H); 3.30–3.80 (m, 84H); 4.00–4.95 (m, 28H); 5.50–6.00 (m, 26H); 7.01–7.08 (m, 2H); 7.23–7.29 (m, 2H); 7.40–7.48 (m, 2H); 7.60–7.70 (m, 2H); 7.90–7.97 (m, 2H); 8.30–8.35 (m, 2H). IR (KBr, cm )1 ): m 3383.3, 2928.2, 2151.4, 1703.7, 1653.6, 1522.2, 1368.4, 1231.9, 1156.0, 1080.2, 1030.5, 945.7, 859.5, 755.9, 706.8, 579.5, 531.7. Elemental analysis calculated (%) for C 112 H 164 O 72 N 6 : C, 48.98; H, 5.98; N, 3.06. Found: C, 48.77; H, 6.12; N, 3.24. Synthesis of metallobridged bis(b-CD)s 2 According to the Liu et al. [16], bis(b-CD)s 1 was added dropwise to a dilute aqueous solution of slightly excess cop- per (II) chlorate in an ice-water bath. Several drops of chlo- roform were further added, and the resultant solution was kept at 5 °C for 2 days. The solution was then evaporated under reduced pressure, and the precipitate formed was collected by filtration, washed successively with a small amount of ethanol and diethyl ether, and dried in vacuo to give complex 2 as a green solid with 63% yield. UV ⁄ vis k max (H 2 O) ⁄ nm (log e): 349.5(1.36). IR (KBr, cm )1 ): m 3419.5, 2930.3, 2048.1, 1637.6, 1536.4, 1406.0, 1337.1, 1301.6, 1238.3, 1155.3, 1121.3, 1078.7, 1028.9, 946.5, 856.1, 755.1, 706.6, 618.1, 579.2, 531.3. Elemental analysis calcu- lated (%) for C 112 H 164 O 72 N 6 Æ2CuCl 2 : C, 44.59; H, 5.44; N, 2.79. Found: C, 44.85; H, 5.72; N, 3.04. Redox iodometric titration of copper (II) was also per- formed to establish the coordination stoichiometry of com- plex 2. We dissolved 1.508 g of complex 2 in 50 mL of ultra-pure water, and added 25.00 mL of the complex 2 solution to a 125 mL Erlenmeyer flask. This was analyzed iodometrically. Copper (II) was first reduced to Cu(I) by KI according to the following reaction: 2Cu 2þ þ 4I À ! 2CuIðsÞþI 2 and the liberated I 2 was titrated against thiosulfate; 26.00 mL of 0.020 m Na 2 S 2 O 3 was required to titrate the liberated I 2 according to the following reaction: I 2 þ 2S 2 O 2À 3 ! 2I À þ S 4 O 2À 6 The percentage of copper in the sample was 4.41. The results confirmed that the mole ratio of complex 2 to B. Tang et al. Glutathione determination using bis(b-CD)s FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS 1515 copper (II) was 1 : 2, which was consist with the Job’ s plot of the 1 ⁄ Cu 2+ system at 349 nm. Calibration graph Into a series of 10-mL colorimetric tube were sequenti- ally added different aliquots of GSH stock solution containing 0–2.00 · 10 )4 molÆL )1 of GSH, 2.00 mL of 2.00 · 10 )4 molÆL )1 metallobridged bis(b-CD)s 2 and 2.00 mL of 0.10 mol Æ L )1 NaCl ⁄ P i (pH 6.00). The mixture was diluted to mark with ultra-pure water, shaken thor- oughly and equilibrated at room temperature for 10 min. The fluorescent intensity of the solution was measured at 365 ⁄ 480 nm (Fig. 7). Acknowledgements This study was supported by the National Basic Research Program of China (973 Program, 2007CB936000), National Natural Science Funds for Distinguished Young Scholar (No.20725518), Major Program of National Natural Science Foundation of China (No.90713019), National Natural Science Foun- dation of China (No.20575036) Important Project of Natural Science Foundation of Shandong Province in China (No.Z2006B09) and the Research Foundation for the Doctoral Program of Ministry of Education (No.20060445002). References 1 Meiser A & Anderson M (1983) Glutathione. Annu Rev Biochem 52, 711–760. 2 Sezginturk MK & Dinckaua E (2004) An amperometric inhibitor biosensor for the determination of reduced glutathione (GSH) without any derivatization in some plants. 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Glutathione determination using bis(b-CD)s FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS 1517 . was developed for the determination of glutathione. The analytical application for glutathione was investigated in NaCl ⁄ P i (pH 6.00) at room temperature. The. from a series of 11 reagent blanks, and S is the slope of the standard curve. The relative standard deviation was 2.5%, obtained from a series of 11 standards