1600 Anal. Chern. 1884, 56, 1600-1603

(34) Zhdanov, S. I , I n “Encyclopedia of Electrochemistry of Elements”; Bard. A. J., Ed.; Plenum: New York, 1975; VoI. 4, Chapter 12.

(35) Nriagu, J. 0.; Coker, R. D.: Kemp, A. L. K. Limnol. Oceanogr. 1979, (41) Schmidt, M.; Sand, T. J . Inorg. Nucl. Chem. 1964, 26, 1173-1177. (42) Iguchi, A. Bull. Chem. SOC. Jpn. 1958, 31, 597-605.

24, 383-389.

RECEIVED for January 30,1984. Accepted April 13,1984. This research Was supported by the U.S. Bureau of Mines under Contract No. JO 100065 to L. M. Cathles and H. L. Barnes

vania State University.

(36) Makhlja, R.; Hltchen, A. Talanta 1978, 25, 70-84. (37) Zezula, I . Chem. Lis@ 1953, 47, 1303-1308. (36) Subrahmanya, R. s. Proc,-Indkn Acad. sci., Sect. A 1955, 42,

267-278.

CRC Press: Boca Ratan, FL, 1977; p 8-94, (39) WeaSt, R. C.3 Ed. ”Handbook Of Chemistry and Physics”, 58th Ed.:

and by the ore it^ ~~~~~~~h section of The pennsyl- (40) Moses, C. 0.; Nordstrom, D. K.; MiUs, A. L., submitted to Talanta.

High-Performance Thin-Layer Chromatography of Gibberellins in Fermentation Broths

Patricia Holt Sackett Abbott Laboratories, 14th Street and Sheridan Road, North Chicago, Illinois 60064

A method has been developed for high-performance thin-layer chromatography (HPTLC) that yields quantitative assay val- ues for gbberellns A, and A, f A,, comblned, In fermentation broths. The llnear ranges for both measurements were from 0 to about 200 pg/mL. Preclslon was 3-4% for repilcates of a single sample, and assay values calculated by two ana- lysts agreed wHhln 6% for A,. Recoveries were sllghtly hlgh for A,. Agreement was good when 14 fermentation broth samples were assayed by both HPTLC and HPLC methods. Resolution from potential interferences, notably glbberellenlc acid, Is documented.

Separation and quantitation of the gibberellin family of plant growth regulators have received much attention in the literature (2-4). The small differences in functional groups between the various derivatives place high demands on the specificity and resolving power of any chromatographic technique. With the advent of high-performance liquid chromatography (HPLC), interest shifted from traditional thin-layer chromatographic (TLC) methods, and focused in- stead on the newer technique. For complex sample matrices (fermentation broths, plant tissues), column fouling could occur, requiring extensive sample cleanup or frequent re- placement of expensive columns. Sample throughout was limited by the run time of a single sample and possible column wash and reequilibration steps between samples.

The recent appearance of 10-pm high-performance thin- layer chromatography (HPTLC) plates, as well as spotting and densitometer instrumentation designed with their use in mind, makes this technique a viable alternative for those without extensive experience with TLC. The increased ef- ficiency of these plates allows separation of more complex mixtures and similar compounds in a shorter time. In this laboratory a method was needed that would allow quantitation of selected gibberellins in as many as 50-100 samples of fermentation broth per week. The following describes a system capable of separating gibberellic acid (AS, 1) from related compounds A4 (2) and A7 (3). Because quantitation of the A3 species, the predominant component in commercial formulations, was deemed of primary importance, separation of the A4 and A7 derivatives was not stressed. Mobile phases capable of separating these closely related species generally contain carcinogenic solvents such as benzene or potentially

I -.

2

3, hazardous combinations such as chloroform + acetone. The mobile phase adopted here does not resolve A4 and A7, and validation work proceeded based on the total combined con- centration of the two.

EXPERIMENTAL SECTION Compounds, Solvents, and Reagents. Reagent grade ma-

terials were used unless otherwise specified. Standards and samples were diluted with methanol containing 0.1 g/2 L 4,5- dihydroxy-3-(p-sulfophenylazo)-2,7-naphtha~enedisulfonic acid (DSNDA) (Eastman Kodak, Rochester, NY) acting as a visual locator. The mobile phase consisted of 4:5 (v/v) cyclohexane (Aldrich Chemical, Milwaukee, WI) + acetone and was stored over 3A molecule sieves (Aldrich). Gibberellins AS, A4, and A7 (Abbott Laboratories, North Chicago, IL) were at least 99% pure; stock solutions were prepared fresh weekly and kept refrigerated when

0003-2700/84/0356-1600$01.50/0 0 1984 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984 1601

not in use. Fresh dilutions of the stock standards were made daily to contain approximately 160 pg/mL A3 and 64 bg/mL each A, and A7.

Chromatographic Plates. HPTLC Silica gel 60 plates (10 X 10 cm) (E. Merck, Darmstadt, G.F.R.) were used. To remove organic interferences, plates were “developed” their full length with methanol and then dried at 105-110 OC for at least 1 h before use. Plates were used within 3 days of washing or were rewashed and redried.

Sample Application and Equilibration. A Camag Linomat I11 (Camag, Muttenz, Switzerland) was used to spray 5 WL of each solution in a 6-mm band at a rate of 6-10 s/pL under a stream of nitrogen. The bandwidth was approximately 0.5 mm. Solutions were applied to opposite edges of the plate with standard in the first (i.e., far left) position on each side. A single standard was used to maximize the number of samples spotted per plate. After being spotted, plates were deactivated by a 15-min exposure in a 72% relative humidity chamber containing concentrated HzS04 + HzO (27:100, v/v). The chamber was supplied with fresh solution daily and allowed to equilibrate overnight before use. To maintain equilibrium, the chamber was exposed to the labo- ratory atmosphere as little as possible during introduction and removal of plates.

Plate Development and Visualization. Both sides of the plate were developed simultaneously in a horizontal development chamber (Camag) with 4:5 cyclohexane/acetone. Development continued until the two solvent fronts met at the center of the plate, requiring about 10-15 min. After air-drying, the plate was placed horizontally in a conditioning tray (Camag) containing concentrated hydrochloric acid. The conditioning tray’s relatively small headspace and the parallel configuration of the plate and solvent surface enable uniform exposure of the entire plate. After 30 min the plate was removed and dried for 10 min at 105-110 OC.

Detection and Quantitation. The fluorescent gibberellins were measured in situ immediately after visualization by a Camag TLC scanner with excitation at 366 nm from a mercury lamp and emission controlled by a 400-nm cutoff fluorescent filter. The scanner was in two-way communication with a Model 3390A integrator (Hewlett-Packard, Palo Alto, CA) via an interface (Camag) allowing an entire side of the plate to be scanned without operator intervention. Integration data for each track were re- ported before the next track was scanned. Instrument settings (10 mm band-pass, 0.5 mm/s scan rate, 3 mm beam length, and 0.3 mm beam width) were kept constant after initial optimization. Assay values for Aa and A4 + A7 were generated by an external standard calculation performed by the integrator. All samples on a given side of the plate were compared to the standard on that side.

RESULTS AND DISCUSSION Merck HPTLC plates (10 X 10 cm) were used after com-

parison with a variety of plates from other sources. Overall the Merck plates were found to be superior in terms of res- olution, uniformity, and the level of background interferences; 10 X 20 cm plates were tried but rejected for routine work when variations were seen in standards spotted on different positions of the plate. The larger plate, with its higher capacity for samples, takes up to 1 h to spot and may be more severely affected by changes in room humidity than the small plates which can be spotted in 15-20 min.

Figure 1 presents the integrator output of standard and a typical fermentation beer sample. The elution characteristics of the components of interest are greatly affected by the amount of time the spotted plate is exposed to 72% relative humidity vapors. If the plate is left in the humidity chamber for only a few minutes, the A3 band is not completely removed from the origin and quantitation suffers. Likewise, if the plate is overexposed to high humidity, the A4 + A, band is pushed into the solvent front, and the high background levels preclude determination of these species.

Assays for gibberellins have traditionally taken advantage of their acid-induced fluorescent properties to provide sensitive and selective methods of quantitation (5). Ethanol/sulfuric

Table I. Rf Values of Selected Gibberellins gibberellin Rf fluorescent color

is0 A,

gibberellenic acid

1-t 3 lactone is0 A,

7-deoxy A,

A3

A4 A,

0.32,

0.40 0.46 0.47 0.72 0.74 0.74 0.74

0.15 purple and yellow/

orange/yellow purple purple purple light blue light green yellow

orange

TableII. Correlation Data for Linearity of A, and A, and A, by HPTLC

A3 A, and A, peak integrator peak integrator area peak area peak

counts height counts height

corr coeff 0.9959 0.9995 0.9988 0.9992 slope 0.00072 0.0023 0.00024 0.00068 Y intercept, 11.4 -6.0 5.4 -4.1

kg/mL X intercept -15713 2640 -22171 5924

acid sprays have been used with large particle size TLC plates, color development hastened by heating on a hot plate until just before the plates char. This method gave poor repro- ducibility on high-performance plates because the relatively large droplets of spray reagent yielded uneven detection of the narrow bands afforded by HPTLC plates. In addition, heating over a hot plate often darkened the plates and in- creased background interference, making densitometry dif- ficult if not impossible. Plates were dipped in Carr-Price reagent (20% SbC13 in chloroform) (6) and dried on a hot plate but sensitivity was fairly low. Much better results were ob- tained when the plates were exposed to concentrated hydro- chloric acid vapors and heated in an oven (7). Besides enabling more uniform exposure of the plate to acid vapors, this method gave greater reproducibility and required fewer manipulations of the plate.

Of the gibberellins that might be present in fermentation samples and interfere with AB, gibberellenic acid was of greatest concern. With the present system it was found that components separated by as little as 0.05 Rf value units could be easily discerned. Of the gibberellins tested (Table I), only 1-3 lactone and 7-deoxy A3 are likely to cause severe inter- ferences, both because of their similar R, values and because of their similar fluorescent colors. In comparison, although Is0 A, elutes close to A7 itself, their different fluorescent colors would enable the analyst to distinguish between the two if both were present.

Linearity data were generated by a recording integrator interfaced with the scanning densitometer and were deter- mined by using both peak area and peak height values gen- erated by the integrator. The linear range extends from ap- proximately 0 to 200 pg/mL for both A3 and total A4 + A,; above this range response drops off. Linear regression data for this region (Table 11) suggest that peak height reports will give slightly better assay results than will calculations using peak areas. All assay values reported below used peak height data and suitable dilutions to bring the samples into the linear range.

Because samples are frequently frozen for as long as 1 week before analysis, their stability under various storage conditions was examined. A pooled sample was divided into seven 5-mL portions and one 40-mL portion before freezing. On successive days one small sample was thawed and diluted for assay; the larger sample was thawed, an aliquot removed, and the re-

1602 ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984

Table 111. Stability of Frozen and Refrigerated Samples

conditions freshly thawed frozen/thawed/frozen sample preparation standard preparation

conditions freshly thawed frozen/thawed/frozen sample preparation standard preparation

a ND. not determined.

A,, d m L day 1 day 2 day 3 day 4 day 5 day 7 day 11

1222 1349 1222 1330 1487 1292 1188 1193 1322 1196 1295 1478 ND' 1273 1222 1312 1217 1334 1458 ND N D

162 167 172 ND ND N D N D

A, and A,, &mL day 1 day 2 day 3 day 4 day 5 day 7 day 11

410 431 456 451 442 447 443 407 440 424 446 436 ND 454 410 430 418 443 430 N D ND 154 144 156 ND N D ND ND

Table IV.

repli- cate 1 2 3 4 5 6 7

mean std dev re1 std dev

Precision Study on Fermentation Broth

A, 2 P g h L A4 and A,, d m L analyst analyst analyst analyst

1 2 1 2

265 267 93 118 261 280 87 116 289 281 90 N D 274 288 86 126 272 279 96 113 27 9 2 97 92 122 268 286 89 132

27 1 283 90 12 1 9.0 9.3 3.5 7.0 3.3% 3.4% 3.9% 5.8%

mainder returned to the freezer for future analyses. An assay preparation was also kept refrigerated and sampled on a daily basis to test the stability of samples after dilution (Table 111). Taking into account day-to-day variations in assay values, no apparent degradation occurred during the c o m e of the study. The method of removing an aliquot from the larger sample solution was also studied. Assay values varied as much as 30% for a single sample in which solids were mixed thoroughly with the solution, allowed to settle under gravity, and centrifuged out before removal of an aliquot. To minimize variations between analysts, all solutions were centrifuged before sam- pling.

To determine the precision of the method, a single large sample of fermentation broth was assayed by two analysts

a b Figure 1. Typical output of scanning densitometer as reported by a recording integrator: (a) A, and A4 + A, standards, (b) fermentation broth sample: (1) A,, (2) A, + A,, (3) solvent front, (4) origin.

(Table IV). Both the standard deviation and the agreement between analysts are good for A,; agreement is slightly worse for the less concentrated A4 + A, although standard deviations are still reasonable. Lower relative standard deviations, on the order of 1-2%, were seen for replicate standard prepa- rations. The increased variability was attributed to inter- ferences from the more complex sample matrix. Recoveries

Table V. Recoveries of A, and A, and A, from Spiked Samples of Fermentation Broth (amt recovered/ (amt found/

g(mL g/mL g/mL amt spiked) theor amt) sample spiked found recovered x 100 x 100

A, blank 656 1 99 773 117 118 102 2 198 875 219 111 102 3 296 970 314 106 102 4 395 1072 416 105 102

av 110 102

A,& blank 62 1 80 138 76 2 160 225 163 3 240 296 234 4 320 373 311

95 97 102 101

98 98 97 98

av 98 98

ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984 1603

applied and where. Double applications to a single location were thus eliminated, and spot shape and unformity could also be better monitored. The dye was not eluted from the origin with this mobile phase and hence did not interfere with quantitation.

Although fluorescent intensity has been reported to be constant for as long as 2 weeks (7), it was found that plates scanned immediately after visualization yielded lower assay results than the same plates scanned 24 h later. As a result, all plates were scanned immediately after development and visualization.

For use on a routine basis for large numbers of samples of fermentation broth, the HPTLC method is superior to HPLC in terms of sample throughput and ease of analysis. Sample filtration need not be as stringent for single-use plates, and simultaneous analysis of multiple samples dramatically lowers the cost per sample. In addition, HPTLC plates allow a visual check of the data: when viewed under long-wavelength UV light, gross comparisons of standards and samples can be easily made to see if the calculated values are sensible. The required instrumentation can be quickly mastered by laboratory per- sonnel, even if unfamiliar with chromatographic techniques.

ACKNOWLEDGMENT The author wishes to thank R. Nair and J. Willoughby for

expert assistance and critical comments, M. Roer and L. Lewis for their help in method development, M. Lombard0 for strategic advice and comparative data, and G. Harris for providing samples.

Registry No. 1, 77-06-5; 2, 468-44-0; 3, 510-75-8; isoA3, 19123-67-2; gibberellenic acid, 546-09-8; isoA7, 2074-38-6.

Table VI. Comparison of Assay Values from HPTLC and HPLC

A, and A,, A,, .uug/mL PdmL fermenta-

tio n broth sampleno. HPTLC HPLC HPTLC HPLC

R355-2 R355-3 R355-5 R355-6 R355-7 R355-8 R355-9 R35 5- 12 R355-13 R355-14 R355-15 R355-16 R355-17 R355-20

1075 936 91 1 938 333 633 925 608 501 789 788 250 449 606

1099 926 843 900 300 634 819 606 593 816 905 265 47 5 580

63 87 6 3 96 75 28 84

164 185

22 17

40 38

< 57

97 96 67

105 91 36 96

139 146

31 28 54 4 3 32

from samples spiked with A, and & + A7 at various levels were slightly high for A3 (Table V).

Fourteen samples of fermentation broth were assayed by the HPTLC method and compared to data obtained from HPLC methods for A3 and A4 + A7 (8) (Table VI). Paired t tests gave correlations of 0.97 and 0.94, respectively, indi- cating a good fit between the two methods ( t = 1.09 for A3 and -0.89 for A4 + A7, p > 0.05). Because conditions were optimized for A3 quantitation, A4 + A, results are less satis- factory and are probably due in part to the lower levels present in the sample.

During method development several minor problems were encountered which severely affected chromatographic quality. Molecular sieves used to dry the mobile phase purchased from one manufacturer leached an unidentified organic component into the mobile phase which increased background noise when the plates were scanned. A yellow band could be seen along the solvent front under white light and the solvent front was fluorescent under long-wavelength UV light. Plates kept in the drying oven for prolonged periods of time were found to absorb organic vapors which likewise increased background noise.

As a convenience to,the chromatographer, 4,5-dihydroxy- 3-(p-sulfophenylazo)-2,7-naphthalenedisulfonic acid was added to the diluent methanol a t a level of 0.005%. A distinct red spot could be seen after standard or sample application, al- lowing the analyst to see which samples had already been

LITERATURE CITED Kagawa, T.; Fukinbara, T.; Sumiki, Y. Agr. Bid . Chem. 1979, 27, 598-599. Kaldewey, H. In ”Thin Layer Chromatography: A Laboratory Handbook”, 2nd ed.; Springer-Verlag: New York, 1969; Chapter 0, pp 479-481. Reeve, D. R.; Crozier, A. In “Isolation of Plant Growth Substances”; Cambridge University Press: CambrMge, 1978; pp 41-78. Lin, J.-T.; Heftmann;,E. J . Chromatrogr. 1981, 213, 507-510. Kaldewey, H. In Thin Layer Chromatography: A Laboratory Handbook, 2nd ed.; Springer-Verlag: New York, 1969; Chapter N. pp 485-487. Segura, R.; Navarro, S. J . Chromatogr. 1981, 217, 329-340. Zhou, L.; Shanfield, H.; Wang, F.-S.; Zlatkis, A. J . Chromatogr. lg81,

Schaar, J. C., Abbott Laboratories, private communication, Oct 29, 1979.

217, 341-348.

RECEIVED for review September 15,1983. Resubmitted and accepted April 4, 1984.