Automation of the Anthrone Assay for Carbohydrate Concentration

Anal. Chem. 2010, 82, 1786–1792

Automation of the Anthrone Assay for Carbohydrate Concentration Determinations Vincent E. Turula, Jr.,* Thomas Gore, Suddham Singh, and Rasappa G. Arumugham Pfizer BioTherapeutics Research and Development, 4300 Oak Park, Sanford, North Carolina 27330 Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity. Recently, polysaccharides have been found to have an expanding basis of medical applications and so are receiving attention in fields such as oxygen transport,1 tissue repair,2 and anticoagulation.3 One such use of polysaccharides is in glycoconjugate vaccines in which the antigenic polysaccharides are covalently attached to carrier proteins for enhanced immunogenicity.4-6 The Prevenar conjugate vaccine has had a significant impact on invasive * To whom correspondence should be addressed. Phone: 919-294-1450. Fax: 919-294-1887. E-mail: [email protected]. (1) Eike, J. H.; Palmer, A. F. Biotechnol. Prog. 2004, 20, 953–962. (2) Francis Suh, J.-K.; Matthew, H. W. T. Biomaterials 2000, 21, 2589–2598. (3) T.Nagumo, T.; Nishino, T. In Polysaccharides in Medicinal Applications; CRC Press: New York, 1996; pp 545-574. (4) Jennings, H. J. Adv. Carbohydr. Chem. Biochem. 1983, 41, 155–208. (5) Jennings, H. J. Sood, R. K. In Neoglycoconjugates: Preparation and Applications; Academic Press: New York, 1994; pp 325-371.

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pneumococcal disease.7,8 Prevenar consists of seven Streptococcus pneumoniae capsular polysaccharide serotypes conjugated to a carrier protein, CRM197, a nontoxic variant of diphtheria toxin.9 Streptococcus pneumoniae is the Gram-positive organism responsible for pneumococcal disease. Second generation pneumococcal vaccines are under development to extend protection beyond the Prevenar serotypes.10,11 Colorimetric assays such as the anthrone assay are used for the quantitation of the total saccharide content in a given lot of polysaccharide or conjugate drug substance. The anthrone assay is a classical colorimetric test developed in the 1940s for quantitation of carbohydrates.12-16 Pneumococcal polysaccharide serotypes are chemically diverse but are of welldefined monosaccharide compositions many of which contain neutral hexoses. The hexose monosaccharides are responsive to the anthrone reagent. For the quantitation of specific serotypes composing the Prevenar vaccine, a test polysaccharide sample is analyzed against a mixture of standard monosaccharides equivalent to the actual occurrence in the respective repeat group of the polysaccharide under test. The polysaccharide test sample and standards are heated at approximately 90 to 100 °C in a concentrated mixture of anthrone in sulfuric acid; this mixture is known as the anthrone reagent. This treatment facilitates both the release, through hydrolysis, of the polysaccharide into its constituent monosaccharides and the covalent binding of the anthrone molecule to the free monosaccharide. The anthrone molecule reacts with an open-form of monosaccharide in acidic conditions to form a blue-green color complex. By matching the standard and test polysaccharide compositions, the linearity of the standard mixture matches the linearity of the test polysaccharide and so enables accurate quantitation; this is evident by the high degree of parallelism between samples and standards. A molar concentration is not determined given that polysaccharide size is of a distribution of molecular weight. (6) Glodblatt, D. J. Med. Microbiol. 1998, 47, 563–567. (7) Darkes, M. J.; Plosker, G. L. Pediatr. Drugs 2002, 4, 609–630. (8) Whitney, C. G.; Farley, M. M.; Hadler, J.; Harrision, L. H.; Bennett, N. M.; Lynfield, R.; Reingold, A.; Cieslak, P. R.; Pilishvili, T.; Jackson, D.; Facklam, R. R.; Jorgensen, J. H.; Schuchat, A. N. Eng. J. Med. 2003, 348, 1737– 1746. (9) Klein, D. L. Microb. Drug Resist. 1995, 1, 49–58. (10) Hausdorff, W. P.; Bryant, J.; Paradiso, P. R.; Siber, G. R. Clin. Infect. Dis. 2000, 30, 100–121. (11) D.Scott, D.; Ruckle, J.; Dar, M.; Baker, S.; Kondoh, H.; Lockhart, S. Pediatrics Int. 2008, 50, 295–299. (12) Dreywood, H. Ind. Eng. Chem. (Anal. Ed.) 1946, 18, 499. (13) Morse, E. E. Anal. Chem. 1947, 19, 1012–1013. (14) Sattler, L.; Zerban, F. W. Science 1948, 27, 207. (15) Morris, D. L. Science 1948, 107, 254–255. (16) Scott, T. A.; Melvin, E. H. Anal. Chem. 1953, 25, 1656–1661. 10.1021/ac902664x  2010 American Chemical Society Published on Web 02/02/2010

For some time the anthrone assay has been performed in its original format for polysaccharide concentration determinations. Traditionally, the hydrolysis reaction is performed in glass tubes, incubated in an aqueous bath, cooled, and measured with a spectrophotometer at λ ) 625 nm. The procedure can be adapted to the standard colorimetric assay format such as in the transfer of the reaction contents to a cuvette-spectrophotometer or to a sipper-spectrophotometer in which the sipper delivers the standards and test samples from the glass tubes into the spectrophotometer via a pressurized transfer line and flow-cell cuvette. Sampling difficulties are inherent in this application, as dilutions from mg/mL to µg/mL concentrations are needed to bring the sample to a quantifiable level relative to the standards. Despite rigorous analyst training and precautions, these difficulties are inherent in manual execution of the traditional form of the anthrone assay. Recent work in the automation of analytical techniques has demonstrated the benefit of robotic formatted assays.17-19 Automation of the anthrone assay began with the scaling down of the assay to a 96-well microtiter plate format for ultimate adaptation to the LHS. Recently, the anthrone and phenol-sulfuric acid assays were configured for a manual microtiter plate format.20-22 Scaledown required changes to the traditional anthrone assay such as changes to the dispensing of liquids, volumes used, mixing procedures, and heating of the plate. However, these anthrone assay conditions stressed the hardware of the LHS and precautions were necessary for successful implementation. Reported herein is the novel adaptation of the anthrone assay to a LHS for the advantage of enhanced accuracy, precision, and improved ease of execution. All of the liquid handling operations were accomplished with an eight-channel automated pipet manifold. Within the LHS system plates were moved from a designated loading space to a heating unit and eventually into the plate reader for measurement. For this work both dextran polysaccharides of various chain lengths and the pneumococcal polysaccharide serotype (PnPs 6A) were used to optimize conditions, format the plate configuration, and assess performance. In this assay format a quantitative determination of the polysaccharide concentration was made from an array of dilutions, to accommodate a range of possible concentrations, with multiple replicates. MATERIALS AND METHODS Liquid Handling System Operation and Data Analysis. For the generation of all data contained in this correspondence, the Perkin-Elmer (Norwalk, CT, U.S.A.) Janus automated workstation equipped with a Victor plate reader was used to monitor absorbance with a fixed wavelength of 620 nm controlled with Wallac 1420 Manager software. Three sizes of PE RoboRack disposable conductive pipet tips with filters were used: 25, 200, and 1000 µL. The entire colorimetric analysis, sample dilutions, hydrolysis, and anthrone complexation reaction were carried out in Becton (17) Rutherford, M. L.; Stinger, T. Curr. Opin. Drug Discovery Dev. 2001, 4, 343–346. (18) Chapman, T. Nature 2003, 421, 661–666. (19) Hawker, C. D. Clin. Lab. Med. 2007, 27, 749–770. (20) Laurentin, A.; Edwards, C. A. Anal. Biochem. 2003, 315, 143–145. (21) Masuko, T.; Minami, A.; Iwasaki, N.; Majima, T.; Nishimuro, S.-I.; Lee, Y.C. Anal. Biochem. 2005, 339, 69–72. (22) Leyva, A.; Quintana, A.; Sa´nchez, M.; Rodriguez, E. N.; Cremata, J.; Sa´nchez, J. C. Biologicals 2008, 36, 134–141.

Dickinson Biosciences Falcon 96-well flat bottom cell culture plates (Franklin Lakes, NJ, U.S.A.). The microtiter plates were moved to specific locations on the Janus deck for unit operations via the Janus robotic arm. To each well 100 µL of either blank, standard, or sample were mixed with 200 µL of anthrone reagent. For quantitative anthrone analysis, the outer plate perimeter positions were not used. Given both the corrosiveness and the high viscosity of the anthrone/sulfuric acid reagent, it was necessary to use a specific mixing procedure, specific in terms of repetitive reciprocating movements at specific heights within the wells. Pipetting, plate movement, and analysis were coordinated with PE WinPrep software. For hydrolysis and color development microtiter plates were incubated with an Inheco incubator shaker system (Munich, Germany) set at 95 °C for 20 min. Raw absorbance values from the Victor plate reader, with Wallac 1420 Manager software, were exported into an Excel spreadsheet for concentration determination. For removal of residual sulfuric acid-anthrone, after each analysis, the pipet manifold was thoroughly rinsed with water. Also, the exterior of the pipet manifold was manually wiped after each analysis. Anthrone Reagent. Both ACS grade (97%) anthrone and concentrated ACS sulfuric acid (95-98%) were also from SigmaAldrich (St. Louis, MO, U.S.A.). The anthrone reagent was prepared by dissolving 1.00 g of anthrone into 500 mL of concentrated sulfuric acid. The reagent was protected from light and was used within 24 h of preparation. Saccharides. Monosaccharides were used to quantitate the polysaccharide test articles. Monosaccharides composing the PnPs 6A repeat group were R-D-glucose anhydrous, 96%, D-galactose, 97%, and L-rhamnose monohydrate, minimum 99%, all from SigmaAldrich. Pneumococcal serotype 6A polysaccharide samples were prepared by Pfizer BioTherapeutics, Sanford, NC, U.S.A. All high purity, 99+% dextran polysaccharide materials, produced at three unique sizes, 215, 850, and 3755 kDa, were from Phenomenex Inc. (Torrance, CA, U.S.A.) and were used as received. Stock solutions were prepared at 5 mg/mL in HPLC grade water and transferred into 1 mL aliquots in single-use vials which were stored at -20 °C until use. To ensure the highest degree of quantitative accuracy, the calibration standard monosaccharide mixtures were matched to a repeat group of the saccharide test sample in the identical molar ratio. The PnPs serotype 6A repeat group is composed of the molar ratio of 1:1:1 galactose to glucose to rhamnose ribitolphosphate, Figure 1 structure of PnPs serotype 6A. Each of these monosaccharides within the standard mixture was prepared at a concentration of 1 mM. For all quantitative analyses of PnPs 6A, a calibration curve was generated by the dilution of the standard mixture into the range of 8, 15, 23, and 30 nanomoles (nM) to a final volume of 100 µL. For dextran quantitation, glucose was used to generate the calibration curve within the same concentration range as PnPs 6A. Given the similar responsiveness of these polysaccharides to anthrone in the microtiter plate format the range from 8 to 30 nM was most appropriate for the quantitation of the test polysaccharide. Both statistical analysis calculations, namely, the analysis of variance with the dextran plate bias experiment and the determination of the confidence intervals within the intermediate precision Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

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Figure 1. Structure of repeat group of PnPs 6A polysaccharide [2)-R-D-Galp-(1f3)-R-D-Glcp-(1f3)-R-L-Rhap-(1f3)-D-ribitol-(5-P-(Of]n. Table 1. Consistency of Plate Absorbances from Three Sizes of Dextran Polysaccharide As Analyzed by LHS-Based Anthrone Are Illustrated by the Mean Absorbance and Percent Relative Standard Deviation (% RSD)a mean absorbance ± %RSD at 620 nm for each dextran size (kDa) replicate plate analysis

215

850

3755

1 2 3 4 mean standard deviation % RSD

0.464 ± 0.0094 0.465 ± 0.0078 0.463 ± 0.0104 0.464 ± 0.0086 0.464 0.001 0.17

0.477 ± 0.0100 0.482 ± 0.0235 0.481 ± 0.0124 0.480 ± 0.0123 0.480 0.002 0.50

0.468 ± 0.0106 0.464 ± 0.0223 0.464 ± 0.0096 0.465 ± 0.0116 0.465 0.002 0.40

a For each dextran sized material, four replicate plate analyses were performed as described in the Methods and Materials section. Into each well of 100 µL of dextran sample, diluted 1:150, 200 µL of anthrone reagent was added. For each mean calculation of each plate all 96 wells were used.

data, were performed using JMP version 6.0.0, Statistical Analysis Software (Cary, NC, U.S.A.). RESULTS Plate Bias. Dextran polysaccharides were analyzed in the entire plate, all of the 96 wells, to assess the uniformity of pipetting, consistency of volume transfer, and the effect of plate position on anthrone response. This was a necessary precursor for formatting the plate for the subsequent quantitative analysis by anthrone; it was essential to determine if all plate positions could be used for the quantitative analysis of polysaccharide samples of unknown concentration. The consistency of dextran response to anthrone was a combination of the LHS volume delivery as well as the plate position. To differentiate, the closeness of plate absorbance as measured by a within plate percent standard deviation was the indicator for LHS volume delivery. Plate position bias was analyzed by analysis of residuals, which is the difference between the grand plate average and each individual position. Three sizes of dextran polysaccharide, at 215, 850, and 3755 kDa, were prepared at approximately 5 mg/mL in water and transferred to the LHS for analysis. The LHS system performed all aspects of the analysis, namely, sample dilution, transfer of aliquots to the entirety of the plate, mixing of sample with the anthrone reagent, hydrolysis, color development, and absorbance measurement; no standards were plated. For each dextran solution four replicate 96-well plates, with entire coverage, were analyzed, and raw absorbances were recorded and used for assessments. Results are tabulated in Table 1. Within plate precision was excellent for all dextran plates indicating uniform anthrone color development in all 96 wells. Analysis of variance for each set of the three sizes of dextran revealed all four replicate plate means were not statistically different. The 850 kDa polysaccharide 1788


solution had a slightly higher mean absorbance, Table 1, 480 absorbance units; this was due to a slightly greater amount of dextran weighed in the preparation. The individual plate absorbances from the dextran plating experiments were tallied and examined for deviations and trends across plate rows and positions, date not included. The consistency of the color development was evident by the closeness of absorbances and the high degree of similarity between each row. Significant outliers or appearance of an edging effect such as a deviation in rows A and H or columns 1 and 12 was not observed. Edging effects are manifested as changes in absorbances in the perimeter positions and are caused by either inconsistent plate heating or plate warping during the 95 °C hydrolysis. The lack of a pronounced edging effect indicated that volume transfers were consistent to all positions and that no significant plate distortion, such as warping, which would alter the absorbance, occurred. The plates were then statistically examined for minor deviations that could diminish the precision and accuracy of the LHS anthrone assay. Positional residuals were calculated from the differences between the absorbances of each of the 96 wells and the grand mean for the entire plate. In Figure 2 the deviation from the grand mean absorbance, residuals are graphed, rows A to H versus the well columns. In this fashion, deviations within the plate were accentuated enabling the identification of positional bias. In Figure 2, two features are noticeable. First, a slight downward deviation with the last three plate columns is apparent in the case of all three dextran polysaccharides. Second, residual analysis shows that row B is generally lower than other rows for all dextran sizes. In general, for all plates the very slight positional effect of both columns and rows trend slightly downward from left to right and from top to bottom. Nonetheless, in these graphs the range

Figure 2. Deviation of each individual well from the grand mean for various sizes of dextran polysaccharides. The differences between each position from the grand mean are graphed versus well position. Row position symbols are indicated in inset A to H in each panel. Panel (A) is of the 215 kDa, panel (B) is of 850 kDa, and panel (C) of 3755 kDa.

of the deviation from the mean is approximately 10% of the mean absorbance; most scatter occurs within ±5% demonstrating that all wells could be used for an accurate anthrone determination. While the dextran analysis showed acceptable performance for the entire plate, for the purposes of quantitation a plate template was programmed such that the outer parameter of 36 wells was excluded. Only 60 interior wells, were used with blanks, calibration standards, and samples.

Quantitative Analysis. In preparation of the anthrone assay, polysaccharide samples require depolymerization via hydrolysis.13,23 This serves to break the polysaccharide into the constituent monosaccharides of its repeat group. Under acidic conditions monosaccharides in the open form react with the anthrone molecule to yield the colored complex that enables quantitation. (23) Ip, C. C.; Manam, V.; Hepler, R.; Hennesey, J. P., Jr. Anal. Biochem. 1992, 201, 343–349.


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Table 2. Accuracy of the LHS-Based Anthrone Analysis of Dextran Is Indicated by the Closeness of the Measured Dextran Concentration and % Accuracya

assay replicate

measured dextran concentration (mg/mL)

accuracy (% of measured to actual)

1 2 3 4 5 6 7 8 9 mean standard deviation % RSD

5.063 4.930 4.955 5.030 4.865 5.396 5.014 5.277 5.125 5.073 0.170 3.4

101 99 99 101 97 108 100 106 102 101

a For this experiment dextran was weighed and prepared to an initial concentration of 5 mg/mL and then further diluted by the LHS. The sample (100 µL) was used in the analysis. Results indicated in this table are back calculated based on the dilution factor. There are nine replicate analyses.

Table 3. Intermediate Precision Values from PnPs 6A Polysaccharide Analysesa

assay

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 mean standard deviation % relative standard deviation upper confidence interval (at 95%) lower confidence interval (at 95%)

PnPs 6A measured standard concentration (mg/mL) deviation

6.511 6.842 6.606 6.857 6.652 6.370 6.484 6.799 6.494 6.727 6.561 6.612 6.392 6.506 6.283 6.454 6.575 6.776 6.536 6.689 6.675 6.382 6.581 0.159 2.4% 6.651 6.510

0.291 0.274 0.202 0.206 0.287 0.297 0.293 0.285 0.173 0.213 0.228 0.292 0.214 0.237 0.139 0.194 0.187 0.172 0.206 0.171 0.355 0.112

a The PnPs 6A polysaccharide was diluted as described in the Results - Quantitative Analysis section. Twenty-two independent assays conducted over 60 days with resulting concentrations and accompanying interassay standard deviation. The mean and 95% confident intervals have been determined for the complete data set.

The responsiveness of the anthrone molecule to polysaccharides is dependent upon several elements of structure. Capsular pneumococcal polysaccharide serotypes exist with a diverse monosaccharide composition including hexoses, uronic acids, and amino sugars as well as differences in the configuration of glycosidic linkages, phosphodiester linkages, and repeat group size;24 all of which impact responsiveness. For this application the pneumo1790


coccal serotype 6A polysaccharide was quantitated against the monosaccharides composing the repeat group, namely galactose glucose and ribitol in the portion to their occurrence in the polysaccharide. A standard microtiter plate template was generated and used for all quantitation experiments. The template was designed to accommodate a polysaccharide test sample within the expected concentration range, with a high-degree of replication. Each blank, standard, and sample dilution was replicated six times. The polysaccharides were diluted, from approximately 5 mg/mL, at ratios of 1:100, 1:75, 1:50, 1:40, and 1:35 with water so that the response would fall within the standard curve range. For plate bias experiments dextran was diluted 1:150. All dilutions contained within the calibration range were used in the determination of the concentration. As such one plate run yielded one concentration for the test sample; with the replication the final concentration was reported as final concentration ±% RSD within the plate. Hydrolysis Optimization. The optimal hydrolysis period was determined by the measurement of the polysaccharide test sample concentration as a function of the incubation time at 95 °C. The optimal hydrolysis period is a balance between the time required to thoroughly hydrolyze the polysaccharide without excess time that causes decomposition of the monosaccharide-anthrone color complex. The PnPs 6A hydrolysis was shown to be consistent for 20 to 30 min yielding very similar results. For all subsequent experiments the hydrolysis period for PnPs 6A was set at 20 min. Hydrolysis periods of less than 20 min generally produced lower and less consistent results, data not included. Additional hydrolysis time caused a slight drop in the polysaccharide concentration indicating monosaccharide decomposition. For all hydrolysis times examined, calibration curve parameters were consistent. Accuracy. The measure of accuracy of the automated anthrone assay was best assessed by the analysis of dextran. The high degree of purity (99+%) of the commercially available dextran, and the accuracy of mass measurement enabled the preparation of the 850 kDa dextran stock solution at exactly 5 mg/mL. The solution was then analyzed as per the standard LHS program and diluted accordingly via the microtiter plate template. For all dextran quantitative analyses the standard calibration curve was produced from glucose monosaccharide. The results from nine unique assays are reported in Table 2. Accuracy was calculated as a percent difference between the measured concentration and the actual concentration of the dextran stock solution. The average accuracy of these nine analyses was determined at 101%, ranging from 97 to 108%, with precision of 3.4% RSD. Intermediate Precision. The reliability of the anthrone assay is most important for the evaluation of commercial production polysaccharide. This is particularly true for processing and for site-to-site transfer. Reliability was assessed by an intermediate precision study in which a single lot of purified PnPs 6A was analyzed over an approximate 2-month period. During this time multiple lots of anthrone reagents, microtiter plates, and calibration lot standards were used on different days. A total of 22 assays were performed and are tabulated, in terms of final concentration and within plate standard deviation, in Table 3. Concentration values reported were generated by all replicate sample dilutions. (24) Kamerling, J. P. In Streptococcus pneumoniae Molecular Biology & Mechanisms of Disease; Mary Ann Liebert, Inc.: Larchmont, NY, 2000; pp 81114.

Table 4. Robustness PnPs 6A Native Polysaccharide Average Concentration Determination after Normal Sample Preparation (Left) and 10% Decrease in Sample Volume for Sample Dilution Preparation (Right)a normal dilution procedure

10% decrease in sample volume

standard dilution factors

PnPs 6A concentration standard preparation (mg/mL)

dilution factors with 10% decrease in sample volume preparation

PnPs 6A concentration with 10% decrease sample volume preparation (mg/mL)

1:100 1:75 1:50 1:40 1:35 average

6.950 6.495 6.592 6.730 6.679 6.689

1:111 1:84 1:56 1:44 1:39 average

6.277 5.946 5.910 6.066 6.040 6.048

a

The standard 200 µL of anthrone reagent was used for these robustness experiments.

Table 5. Robustness of PnPs 6A Native Polysaccharide Concentration Determination after Normal Sample Preparation (Left) and a 10% Increase in Sample Volume for Sample Dilution Preparation (Right)a normal dilution procedure

10% increase in sample volume



dilution factors with 10% increase in sample volume preparation

PnPs 6A concentration with 10% increase in sample volume preparation (mg/mL)

1:100 1:75 1:50 1:40 1:35 Concentration

7.145 6.448 6.231 6.855 6.699 6.675

1:91 1:68 1:46 1:36 1:32 Concentration

7.450 6.872 6.833 7.071 6.922 7.030

a

Final concentrations are indicated below for each. The standard 200 µL of anthrone reagent was used for these robustness experiments.

Table 6. 6A Native Polysaccharide Concentration Determination after Normal Sample Preparation (Left) Is Compared to Preparations with a 10% Decrease (Center) and Increase in the Anthrone Volume Added for Hydrolysis (Right)a normal dilution procedure

10% change in sample volume



PnPs 6A concentration 10% decrease anthrone volume added (mg/mL)

PnPs 6A concentration 10% increase anthrone volume added (mg/mL)

1:100 1:75 1:50 1:40 1:35 average

6.456 6.196 6.380 6.485 6.393 6.382

6.616 6.316 6.432 6.532 6.305 6.440

6.716 6.407 6.047 6.500 6.436 6.421

a

Final concentrations and average are indicated for each.

By employing the replication strategy the within plate, a low variability was maintained, and the polysaccharide concentration precise. The overall % RSD for the entire set of data was 2.4% with a range from 6.283 to 6.857 and a 95% confidence interval of 6.651 to 6.510 mg/mL. Compared to the internally validated manual procedure, in which intermediate precision was measured at 6% RSD, there was an obvious improvement in using the LHS. These experiments legitimized the LHS-based anthrone plate analysis. Robustness. Purposeful deviations of volume deliveries were programmed into the LHS to assess how the LHS-based anthrone assay format tolerated such deviations. For these experiments, the LHS transferred sample volume deviations of 10% above and below the specified dilution factors; standards were prepared as usual. Table 4 compares the deviation of 10% below, and Table 5 is of those with 10% above the standard sample dilution. The dilution factors were adjusted to account for the deviations in both tables and corresponding concentration indicated. For the plate

prepared with 10% less sample the corresponding concentration was 6.048 mg/mL, which compared to the standard preparation, run immediately after of 6.689 mg/mL, showed at 9.6% decreased concentration. Similarly, for the plate prepared with 10% more sample, the corresponding concentration was 7.030 mg/mL compared to the standard preparation of 6.675 mg/mL which yielded an approximate 5.3% increase in measured concentration. While the increased level of sample did not increase the measured concentration by proportional 10%, it did alter the measured concentration to a significantly higher level. Additionally, the LHS was programmed to deliver anthrone at 10% deviations above and below the standard anthrone delivery volume of 200 µL. For these experiments the proscribed 100 µL aliquot volume was added to both standard and sample dilutions. Results are indicated in Table 6 with the specific results per dilution. The variation of the volume of the anthrone effectively changed the path length in each well but did not distort the absorbance consistency. This is illustrated by the closeness of Analytical Chemistry, Vol. 82, No. 5, March 1, 2010

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the three determinations with 180, 200, and 220 µL anthrone with a % RSD of approximately 0.5%. With additional anthrone volume the plate wells were near capacity. DISCUSSION Experiments detailed herein demonstrate that the anthrone assay can be successfully adapted to a commercial LHS. While the traditional execution of the assay is performed in glass tubes with spectrophotometer detection, the assay has been successfully performed manually in microtiter plates21 and here automated in microtiter plates. The viscous concentrated sulfuric acid medium makes liquid transfers prone to inconsistencies; the LHS diminished the likelihood of such transfer errors because of consistent execution. However, in using the LHS for this purpose rinsing steps were necessary to prevent component corrosion from the anthrone reagent. The pipet manifold with the Janus LHS is an eight-channel system; each channel was thoroughly rinsed with water upon completion of the analysis to eliminate any residual sulfuric acid. In the experience of these authors, it is noted that not all commercial LHS systems can reliably perform the anthrone assay because of component susceptibility to acid corrosion from the anthrone reagent. Moreover, in the development of this work it was necessary to optimize the mixing program in terms of plate depth, draw-speed, and repetition of draw-up eject cycles. As noted previously by Leyva et al.22 and confirmed here, the incubation temperature used for the hydrolysis step did not have any deleterious effect on the plate. If plate distortion occurred the plate bias experiments would have detected it. Also, with these experiments edging effects, which might have been caused by poor heat transfer to the well locations at the perimeter, were not observed either. For routine analysis the incorporation of control samples of known concentration, such as dextran, on the plate parameter could be used to assess assay validity for unknown concentration determinations. Any volume transfer issues or plate

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defects could be readily detected by out of trend results. In this case the LHS pipet channels could be recalibrated, and the LHS restarted. In the execution of the LHS-based procedure the plate was prepared, the polysaccharide samples were mixed, then hydrolyzed to depolymerize the polysaccharides into constituent monosaccharides that are reactive to the anthrone molecule, and analyzed. The advantage of a LHS-based microtiter plate format anthrone assay is the consistent and uniform execution, which improved accuracy and precision over manual performance. Additionally, in our laboratories anthrone assay cycle time was improved over manual use. The LHS could complete the analysis in approximately 2 h, whereas an analyst must perform all operations manually. This requires analyst attention for an additional period of time to perform all aspects of the anthrone assay. The opportunity cost for execution of the anthrone assay favor LHS execution over the analyst. Work continues on the analysis of additional PnPs serotypes and application to other polysaccharide molecules of interest. ACKNOWLEDGMENT The authors acknowledge Pat Rolston of Perkin-Elmer for assistance with Janus setup and programming and Miguel Maccio and Bart Zoltan of Pfizer Biomedical Engineering for useful suggestions regarding automation operations. Thanks are extended to Pfizer employees Shaune’ Walters for the generation of feasibility data and Eric Finley and Michelle Turula for the thorough proofreading of this manuscript. Also, Brian Nunnally of Pfizer is acknowledged for his statistical analysis and insights.

Received for review October 21, 2009. Accepted January 19, 2010. AC902664X

Automation of the Anthrone Assay for Carbohydrate Concentration

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