Technical Notes Anal. Chem. 1995, 67, 1290-1292
Determination of Maltodextrin in Psyllium-Based Bulk Laxatives by in Situ Silylation and Supercritical Fluid Chromatography Thomas L. Chester* and David P. Innis The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 538707, Cincinnati, Ohio 45253-8707
Maltodextrin is not easily extracted from psyllium-based products by direct means. In situ silylation of product samples was used to change the dissolution properties of the components and make extraction possible. The concentration of silylated maltodextrin was then determined using open-tubular supercritical fluid chromatographywith external standards, direct injection, and flameionization detection. Maltodextrin is a mixture of glucose oligomers connected almost exclusively with a-(1-4) linkages. It is made by the partial hydrolysis of starch. The degree of polymerization (DP) of individual maltodextrin components varies among different maltodextrins, depending on their processing, but is often in the range of 2-25. Maltodextrin can be measured easily by several liquid chromatographic technique^.'-^ For example, high-performance anion-exchange chromatography, combined with pulsed amperometric detection, offers a combination of speed, sensitivity, and elution range reaching to DP 45.j However, none of these techniques will work unless the components of interest can be dissolved in a solution suitable for injection. Psyllium is crushed seed of Plantago psyllium. It is a valuable dietary supplement for individuals requiring additional fiber. Psyllium is very hydrophilic and, when placed in water, swells into a mucilaginous gum. The flow and dissolution properties of psyllium-based products can be modified by processing with maltodextrin. The determination of maltodextrin in the presence of psyllium is complicated by the dissolution properties of these two components. Maltodextrin is very water soluble but is not highly soluble in other common solvents. Wet psyllium gum is high in both carbohydrate and water content. Thus it provides a strong reservoir for maltodextrin. Therein lies the analysis problem: maltodextrin can only be dissolved well in solvents,
water in particular, which swell psyllium into an unextractable mass. We approached this problem by seeking a discontinuous change in this seemingly intractable behavior. We postulated that making the maltodextrin hydrophobic, through in situ silylation, would allow it to be extracted with a solvent that does not swell the psyllium. The silylation would also derivatize at least some of the hydroxyl groups available in the psyllium carbohydrates, thereby making the psyllium more hydrophobic. Therefore, the swelling and extraction behavior of derivatized psyllium had to be determined experimentally. Silylated maltodextrin, once separated from the psyllium, could possibly be hydrolyzed and determined by one of the liquid chromatographic methods mentioned earlier. However, this would involve considerably more sample handling and would introduce opportunities for errors in the additional steps. Instead, we chose to directly analyze the silylated extracts using opentubular supercritical fluid chromatography (SFC) .‘js7
EXPERIMENTAL SECTION The SFC was a home-built instrument6s7equipped with a roomtemperature, O.O&pL internal loop injector (Model ECI4W.06, VICI, Houston, TX), a 50-pm-i.d. waste-port restrictor, a 2-m x 50-pm4.d. fused-silica retention gap (with the first 50 cm at room temperature), a 10-m x 50-pm4.d. SB-Octyl-50 open-tubular column with a 0.25-pm film thickness (Dionex, Salt Lake City, UT), a robot-pulled tapered capillary restrictol.8 as the column-todetector interface, and a flame-ionization detector (FID). The mobile phase, SFC-grade carbon dioxide (Scott Specialty Gases, Plumsteadville, PA), was used without further purification. Data analysis was performed with a Nelson Turbochrom system (P. E. Nelson, Cupertino, CA) with peaks autointegrated. Direct injections were made from the injection valve onto the retention gapg
(1) Cheetam, C. A.; Teng G. J Chromatogr. 1 9 8 4 , 3 3 6 , 161-172.
(2) Koizumi, IC;Utamura, T.; Okada, Y. J Chromatogr. 1985, 321, 145-157. (3) Hardy, M. R; Townsend, R R Proc. Natl. Acad. Sci. U.S.A. 1988,85,32893293. (4) Townsend, R R; Hxdy, M. R; Hindsgaui, 0.; Lee, Y. C. Anal. Bioclrem. 1988, 174, 459-470. (5) Olechno, J. D.; Carter, S. R; Edwards, W. T.; Gillen, D. G. Am. Biotechnol. Lab. 1987, 5, 38-50.
1290 Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
Chester, T. L.; Innis, D. P. J. High Resolut. Chromatogr. Chromatogr. Commun. 1986, 9, 209-212. Chester, T. L.; Pinkston, J. D.:Owens, G. D. Carbohydr. Res. 1989, 194, 273-279. Chester, T. L.; Innis. D. P.;Owens, G. D. Anal. Chem. 1985, 57, 22432247. Chester, T. L.; Innis, D. P. J. Microcolumn Sep. 1993, 5. 261-273. 0003-2700/95/0367-1290$9.00/0 0 1995 American Chemical Society
n
Table 1. SFC Conditions
oven FID
pressure
110 "C 350 "C hold at 100 atm for 5 min, ramp from 100 to 140 atm in 5 min, 140 to 350 atm in 70 min
using the waste-port-restriction technique to load the valve loop.1o The parameter settings are listed in Table 1. Maltodextrin (Maltrin M-100) was obtained from Grain Processing Corp. (Muscatine, IA). Psyllium (LE-sized mucilloid) and several commercial products, purchased from retail stores, were obtained from The Procter & Gamble Co., Health Care Product Development Division. The commercial products were thoroughly mixed before analysis. In addition, fractions of these products not passing through 100-mesh screens were separately analyzed. Nitrogen was used to purge and ensure dryness of 5mL ReactiVials (Pierce, Rockford, IL) equipped with PTFE stirring bars and air-tight PTFE-lined caps. For each sample of the commercial products and of the sieved fractions, a 50-mg portion was transferred into a vial. To the vial was then added 2 mL of reagentgrade pyridine a. T. Baker, Phillipsburg, NJ), previously dried by saturating with KOH. (Caution: Handle all liquids for this procedure in a suitable hood.) After brief stirring to disperse the solids, 400 p L of (trimethylsily1)imidazole CTMSI) (Pierce) was added, and then the vial was immediately capped." Maltodextrin standards were prepared in psyllium and were similarly derivatized. Each standard contained 32 mg of psyllium (the estimated amount in each product preparation) with 0, 5, 10, or 20 mg of maltodextrin added. All vials were then stirred on a magnetic stirrer at room temperature (-27 "C) for 136 h with occasional slight tipping and rotating of the vials to rinse down the sides. If small lumps formed, the sample vials were placed in an ultrasonic bath for 1-2 min, and then stirring was resumed. The psyllium is not dissolved in this procedure. However, the completeness of derivatization of the maltodextrin was checked in the course of developing this procedure by analyzing standards as a function of time, as described in the next section. After derivatization was completed, each sample solution was drawn into a 1GmL gas-tight syringe through a 25"-diameter syringe filter (Acrodisc CR F'TFE, 0.45 pm, Gelman Sciences, Ann Arbor, MI) attached to a 2-in. syringe needle. After the syringe needle and filter were removed, sample solutions were expelled into 1-dram vials (previously purged with nitrogen) and capped with PTFE-lined caps. Between samples, the syringe and needle were rinsed (in order) with pyridine, acetone, and methylene chloride (all reagent-grade, from J. T. Baker) and then dried with nitrogen. A new syringe filter was used with each sample and was purged with nitrogen just prior to use. The filtered samples were then injected onto the SFC with no additional preparation. After each injection, the syringe and the sample and waste flow path through the injection valve were rinsed (in order) with pyridine, acetone, and methylene chloride. The syringe was then dried in a nitrogen stream, and the valve was flushed with air (through the syringe port). The waste-port restrictor still occasionally plugged at its outlet during this work, (10) Chester, T. L.; Innis, D. P. J Microcolumn Sep. 1989,I , 230-233. (11) Brittain, G. D.;Schewe, L. In Recent Advances in Gas Chromatografihy; Domsb, I. I., Perry, J. A, Eds.; Marcel Dekker, Inc.: New York, NY,1971.
0
time
54 min
Figure 1. Typical SFC-FID chromatograms of derivatized extracts of (a) psyllium, (b) maltodextrin standard in psyllium, and (c) commercial product.
even with this extensive rinsing and drying. Cutting several millimeters of length from the waste-port restrictor outlet was all that was necessary to restore the flow. Silylated maltodextrin elutes as anomer peak pairs in SFC7 ( h o m e r s are diastereomers differing only in the relative position of a hydrogen and hydroxyl group about one carbon.) Quantitation was accomplished using the external standards described earlier. The relative area ratios of the maltodextrin peaks within the chromatograms did not signilicantly vary among the samples and standards. That is, the identity of the specilic maltodextrins in the products could not be distinguished from Maltrin M-100. Quantitation was then performed, for convenience, using only the height of the second anomer peak of the maltoheptaose pair. This was the tallest peak of all the maltodextrin peaks and was located in a very clean area of the psyllium blank. RESULTS AND DISCUSSION Maltodextrin was effectively derivatized and dissolved using this procedure. Separate experiments involving sampling and analysis of maltodextrin derivatives both with and without psyllium present indicated that derivatization was completed in approximately 75 h. The amount of TMSI added to the samples was approximately a 2:l molar excess of that estimated to derivatize all of the hydroxyl groups present, calculated assuming that the entire sample mass was glucose. (Psyllium and maltodextrin both have fewer hydroxyl groups per unit mass than glucose.) We observed that larger amounts of TMSI decreased the time required to reach equilibrium but increased the likelihood of plugging the waste-port restrictor. We also achieved better success at derivatizing and dissolving the maltodexttin in some of the commercial products by performing the derivatization at room temperature for many hours rather Analytical Chemistry, Vol. 67,No. 7,April 1, 1995
1291
Table 2. Analysis Results for the Commercial Product Samples
samplea
maltodextrin concnb
la
44.2 f 2.3 (n = 4, same day) 47 i 8 (n = 8 , 6 days) 44.2 i 1.0 (n = 4, same day) 52 (52.6, 50.5) 53 (53.5, 52.0) 47 (47.1, 47.9) 40 (40.3, 39.2) 33 (34.4, 30.7) 35 (35.4, 33.8)
lb 2a 2b 3a 3b 4a 4b
a Each of four commercial samples, 1-4 were prepared in duplicate, as denoted by the a or b suffix. Determined in the commercial sample as mass percent. Mean, standard deviation, and number of determinations are shown for samples l a and lb. The results of the study involving injections of l a over a &day period are shown separately. The remaining preparations were injected in duplicate. The means of these paired determinations are given (with individual results shown in parentheses).
than at an elevated temperature for a shorter duration. This may have been due to the presence of additional materials, not as soluble in hot pyridine as in room-temperature pyridine. This marginally soluble material, if not dissolved, apparently provided a physical barrier preventing some fraction of the maltodextrin from contacting the silylating reagent. The psyllium changed in appearance somewhat and may have been swollen slightly by the derivatization process. After many hours, some of the psyllium particles would loosely aggregate. However, a gum was not formed, nor was the psyllium appreciably solubilized by the process. A chromatogram of derivatized psyllium extract (with no maltodextrin added) is shown in Figure la.
1292 Analytical Chemistry, Vol. 67,No. 7, April 1, 1995
Typical chromatograms of a silylated maltodextrin standard prepared in psyllium and a commercial product preparation are shown in Figure lb,c. We found maltodextrin at levels ranging from 33%to 53%in the commercial products (samples 1 and 3) and their sieved fractions (samples 2 and 4, respectively), summarized in Table 2. Reproducibility of the entire method was checked by performing two complete preparations of every sample (suffixed a and b in Table 2) and by performing multiple chromatographic analyses of each of these preparations. Preparations l a and l b were analyzed four times each, while the remaining sample preparations were analyzed in duplicate. In addition, preparation l a was analyzed four more times, spanning a total of 6 days. Good agreement was generally realized among the analyses of any particular sample preparation. The large increase in the standard deviation for the &day results of sample l a compared to thesameday results suggests that the analyses should be done over a short time frame. This is often desirable with silyl derivatives. This work establishes the concept of in situ derivatization to change solubility and extraction properties of maltodextrin in psyllium-based products. Further optimization of the procedures may help improve the precision. The use of an autosampler to shorten the total time required for the SFC analyses (by allowing around-the-clock injections) may also help improve precision. Received for review August 23, 1994. Accepted January 17, 1995.@ AC940837A
@
Abstract published in Advance ACS Abstracts, February 15, 1995.
