- Email: [email protected]
Colorimetric determination of macrolide antibiotics using ferric ion
•
:
•
" -.'i.'"~"
,
r~
:
',"?!
Talanta ELSEVIER
Talanta 42 (1995) 1425-1432
Colorimetric determination of macrolide antibiotics using ferric ion Patricia A. Gallagher, Neil D. Danielson
*
Department o1 Chemistry, Miami Unit,ersity, Oxfbrd, OH 45056, USA
Received 8 December 1994; revised 13 March 1995; accepted 14 March 1995
Abstract
Macrolide antibiotics such as erythromycin, oleandomycin, spiromycin, and tylosin are found to react with Fe 3+ in the presence of an acetic acid-sulfuric acid mixture to form a colored product having a useful absorption band at 592 nm. Troleandomycin forms only a weakly colored product upon reaction. The molar absorptivity is about 29001mol -~ c m - ' for erythromycin and the detection limit is 5 lag ml-~. This colorimetric method permits the analysis of fermentation broths containing either erythromycin or tylosin without a separation step.
1. Introduction
Macrolide antibiotics, all composed of either a 14-membered or a 16-membered oxygenated ring with several sugars attached, have proved to be effective antibacterial agents for m a n y years. The structures of five c o m m o n macrolide antibiotics are shown in Fig. I. Erythromycin is certainly the most well-known compound of this class; however troleandomycin is also available as a pharmaceutical product and tylosin is used widely by veterinarians [1]. At least six colorimetric methods have previously been developed for several of these macrolide antibiotics. Hydrolysis of the sugar groups o f erythromycin [2] or oleandomycin [3] in concentrated sulfuric acid produced a yellow product measurable at 470 nm. This reaction has recently been extended to the other three macrolide antibiotics shown in Fig. l with a comparison o f linearity and detection limits [l]. Tetrazolium blue can react generally with reducing agents such as erythromycin to form a dark blue solution [4]. Complex formation with * Corresponding author.
Bromcresol Purple has been applied to the colorimetric determination of erythromycin [5]. A variety of other dyes which undergo ion pair formation with erythromycin in chlorinated solvents can be utilized [6]. Erythromycin and tylosin have been determined at 842 nm using tetracyanoquinodimethane [7]. A coiorimetric method at 532 nm for oleandomycin using diazotized sulphanilic acid is known [8]. However, all of these methods are nonspecific or require a nonaqueous solvent and would be difficult to use for the determination o f macrolide antibiotics in aqueous complicated matrices such as fermentation broths without a prior separation step. The reaction of cis-aconitic anhydride with a tertiary amine such as a macrolide antibiotic permits the formation of a colored product detectable at 525 nm. Although this reaction has been applied to the determination o f tylosin in fermentation broths, extraction of the broth with sufficient isopropyl acetate to give a residue that could be reconstituted to attain a ! m M concentration level is required [9]. We report here a coiorimetric method for macrolide antibiotics which involves the
0039-9140/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0039-9140(95)01587-6
1426
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-/432 o
,,,=,-" "o. -'-F -o~..~-=,-,=,
ERYTHROMYCIN A
=1
Ih
Ih
m=
o~,
n
o
,,~"~?-"'r" "'o3.~g=,,
OLEANDOMYCIN TROLEANDOMYCIN
./
SPIRAMYCIN
~'~
=,
=-u =--¢o0~
~=~
.%=,
== I
O
TYLOSIN Fig. 1. Structures of macrolide antibiotics.
macrolide ring and not the sugar or tertiary nitrogen moiety. After dehydration of the macrolide antibiotic in concentrated acetic acid, the addition of Fe 3+ will generate a coloured product measurable at 592 nm. The determination of erythromycin or tylosin in fermentation broths is possible without sample pretreatment.
2. Experimental 2.1. Chemicals
The macrolide antibiotics (Fig. 1) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The erythromycin ethylsuccinate and sulfisoxazole acetyl oral suspension was
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432
obtained from Abbott Laboratories (North Chicago, IL, USA). There were two different fermentation broths used for this research. One was a dehydrated Antibiotic Medium 3 purchased from Difco Laboratories (Detroit, MI, USA) and the second, donated by Eli Lilly (Indianapolis, IN, USA) was a dehydrated fermentation broth used for tylosin production. The Difco product was composed of beef extract, yeast extract, peptone, dextrose, sodium chloride, dipotassium phosphate, and monopotassium phosphate. The Eli Lilly sample was made up of yellow corn meal, fish meal, corn gluten meal, sodium chloride, ammonium phosphate, calcium carbonate, crude soybean oil, and cane molasses. The ferric chloride was purchased from MCB Manufacturing Chemists, Inc. (Cincinnati, OH, USA). The sulfuric acid and acetic acid were both purchased from Fisher Scientific (Cincinnati, OH, USA) and the triply distilled water obtained from a Barnstead Nanopure distillation unit (Sybron/Barnstead, Dubuque, IA, USA). 2.2. Equipment The spectrophotometry was conducted either on a Varian (Humboldt, CA, USA) DMS 90 UV-ViS spectrophotometer or a Hewlett-Packard (Palo Alto, CA, USA) model 8452A diodearray instrument. The sample compartment was heated to 50°C using a Fisher Scientific (Cincinnati, OH, USA) model 900 isotemp refrigerated circulator water bath. 2.3. Procedure A 500 lag ml -j erythromycin stock solution prepared in glacial acetic acid was allowed to react at 45"C for at least 30 min (often 2h) before use. A ferric ion stock solution was prepared by weighing 0.080 g of ferric chloride, pipeting 2 ml concentrated sulfuric acid and 2 ml of triply distilled water into the 100 ml volumetric flask, and diluting to the mark with concentrated glacial acetic acid. Erythromycin-ferric ion standards generally ranging from 10-250 ug ml- ~ were prepared by pipeting 5 ml of ferric ion stock solution and the appropriate amount of erythromycin stock solution before dilution to a 10 ml volume with glacial acetic acid. The fermentation broths were prepared by adding either 17.5 g of dehydrated Antibiotic Medium 3 or the entire Eli Lilly sample to a 1000 ml volumetric flask and
1427
filling to the mark with triply distilled water. A 5 ml aliquot of the spiked fermentation broth sample was placed in a 50 ml volumetric flask and filled to the mark with concentrated glacial acetic acid. The blank was prepared in exactly the same way as the sample except that the fermentation broth was not spiked with the macrolide antibiotic. All fermentation samples and blanks were heated in the 45°C water bath. The standards for this part were prepared exactly the same as for the sample except triply distilled water was used instead of the fermentation broth. The pharmaceutical sample was prepared by measuring out in a volumetric test tube 1 ml of the erythromycin ethylsuccinatesulfisoxazole acetyl oral suspension and quantitatively transfering it to a 100 ml volumetric flask before filling to the mark with concentrated glacial acetic acid. This was heated in the 45"C water bath. The above procedure for preparing the erythromycin-ferric ion standards was carried out for all the samples. All standards, samples and blanks, after mixing with ferric ion, were allowed to react in the heated (50"C) curvette holder for 15 min before the absorbance measurement was taken.
3. Results and discussion
3.1. Proposed reaction mechanism Previously, it had been proposed [10] that the reaction of cholesterol in an acetic acidsulfuric acid mixture with Fe 3÷ will lead to the formation of trienylic and tetraenylic cations detectable at about 480 nm and 560 nm, respectively (Fig. 2). The wavelength maxima are dependent on the sulfuric acid concentration in the Fe 3+ stock solution. It is also known [11] that erythromycin in the presence of concentrated acetic acid can undergo dehydration, forming the 8,9-anhydroerythromycin-6,9hemiketal (Fig. 3). The hydroxy group beta to the double bond in this hemiketal product is a remarkably similar key functional group to that in cholesterol. Using LC-MS, we confirmed the formation of this hemiketal product as evidenced by the disappearance of the M+,MH ÷ ion peak pair at M/z = 734, 735 and the formation of the ion peak at M/z = 716. No peak for the diene carbonium ion was evident. A peak at M/z = 558 present in both the erythromycin and hemiketal spectra represents the fragment ion of erythromycin minus a
1428
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432
~L
HOAc/H2S04
~L
Fe3+
~
'll" Fe2+
3.2. Optimization of colorimetric response
~tion
Fig. 2. Proposed mechanism of the eholesterol-Fe 3÷ reaction (from Ref. [10]).
sugar group. An absorption spectrum of the hemiketal product after reaction with Fe3+using the optimum conditions shows two strong peaks at 484 and 592 nm (Fig. 4). A spectrum of the cholesterol reaction run using an 80% acetic acid-20% sulfuric acid Fe 3+ solution showed three peaks with wavelength
N(CH3)2
H3'~
I~Cl-t3,0...L.~m/~.~
./~-o43 ~,o,.,~% "sC~ b v , k . ~
maxima at 620, 453, and 412 nm. A spectrum of the hemiketal product run using the same 80% acetic acid-20% sulfuric acid Fe 3+ solution also showed three peaks with wavelength maxima of 596, 486, and 412 nm. A glucose stock solution gave no reaction with Fe 3+, providing furthur evidence that the likely source of the colored product was from the macrolide ring and not the sugar groups attached to the macrolide ring. Although L C MS could not provide a clear mass spectrum for the formation of trienylic or tetraenylic cations for the Fe3+-erthyromycin reaction, we feel this is the likely type of colored product.
,,_L~oN
H3C 0(343 HOAc N(C83)2
•
Fig. 3. Acetic acid degradation of erythromycin A to form the hemiketal product.
A time study of the dehydration reaction in concentrated acetic acid at 45°C for all five compounds shown in Fig. 1 was carried out (Fig. 5). All the macrolide antibiotics except troleandomycin seemed to undergo significant dehydration to form a product that would react with Fe 3+ ion. Because troleandomycin has an ester group instead of the hydroxyl group as in oleandomycin, this difference in reactivity is not surprising. Both spiramycin and tylosin already have some conjugation in their structures which is apparently expanded after reaction with Fe 3+. The spectra of the Fe3+-antibiotic products for oleandomycin, spiromycin, and tylosin were similar to that of erythromycin. The following optimum heating times for the macrolide antibiotic stock solutions were 120 min for erythromycin, 90 min for oleanomycin, 60 min for spiromycin and 0 min for tylosin. Although the optimum heating times were quite varied, in general a reaction time of only 30 rain would be sufficient for good dehydration to the Fe 3+ reactive product. The effect of water in the erythromycin stock solution on the colorimetric reaction was studied (Fig. 6). It appears that 20% water can lower the absorbance to zero; only 5% water in the acetic acid solution caused a 50% decrease in the absorbance of the Fe3+-hemiketal product. Therefore, it was decided that for best detectability the macrolide antibiotic stock solution in acetic acid should be a prepared if possible from a solid instead of an aqueous sample. The proper amount of sulfuric acid needed to be added to the ferric chloride stock solution prepared in glacial acetic acid to give maxi-
1429
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432 1.5001
1.2193
J
0.9385 4C m
$ us m ,i[
0.8577
0.3769
O.Og60
.
400
.
.
.
.
. . 500
.
.
.
. 600
1 700
. . . .
~0
"
Wavelength (am) Fig. 4. Absorbance spectrum of the Fe3+-hemiketal reaction product of erythromycin (250 pg ml-t).
mum absorbance was determined. Ferric chloride solutions containing 1, 2 and 4 ml o f sulfuric acid were prepared and time studies were completed (Fig. 7). In comparison, the cholesterol-Fe 3+ reaction would not work using such low concentrations o f sulfuric acid. The absorbance profiles for the 2 and 4 ml solutions were almost exactly the same. There were some reproducibility problems; the inconsistency in the results were believed to be caused by the sulfuric acid absorbing water. To alleviate this problem, small amounts o f water were added to the ferric chloride stock
solution (Table 1). Therefore, it was determined that 2 ml o f water as well as 2 ml o f sulfuric acid should be added to 100ml o f ferric chloride solution to obtain the optimum absorbance and reproducibility. At room temperature, the F e 3 + - a n h y d r o hemiketal macrolide reaction rate is very slow; therefore, the solution was heated at 40, 50 and 60°C (Fig. 8). At 50°C, the data obtained produced a reproducible plateau from approximately 10 to 30 min. Some loss in signal was observed after 10rain using a 60°C reaction temperature. Therefore reaction con-
OAt 1.2 m
et
•
0.S
o
1.o n
•
m
6
.
m
i
0.4
0.8'
•~
0.I'
fl
<
O. 4
"
[ • Ery~
I " T'."
II
(3.414N I 10414)
l
I • TmlealldOiyCia (3.0"71 • IO"+bl}J • • .+ I • Slmmmymn (2.903 • 10 M ) 1
O.,"
O.0'
•
•
(~L729 x IO'ald ,
I
•
"~
0.3-
~
0.|"
m
8
0.1
II
O.O
o T i m e Sample Stock Solution w a s betted (rain)
Fig. 5. Reaction of macrolide antibiotics with acetic acid at 45°C as a function of time. The reaction was followedat 592 nm using Fe~+.
,o mL of water per
~o 100 mL of Ery~'omycin
3o Solution
Fig. 6. Effect of water in the erythromyein stock solution on the absorbance. The reaction was followed at 592 nm using Fe "~÷.
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432
1430
mined to be 2.5 ppm of erythromycin at a wavelength of 484 nm; however, the linearity at 484 nm was not as good as at the 592 nm. The RSD of the slope at 484 nm was 2.5%.
3000"
I I I I I V h Ill l I O N I ~ ' ~ , h
-_=
•m••a
2000" >,
1000'
a I
~ ,a
<
~ u aaD
/ n R.T., lmL acid [ • R.T., 2mL acid ] [ • R.T., 4mLacidl
0
o
20
40
60
80
i 100
Time (rain) Fig. 7, Optimization of the molar absorptivity as a function of sulfuric acid volume in the Fe 3+-acetic acid solution. The reaction was followed at room temperature (RT) at 592 n m using Fe 3+.
ditions of 15 min at 50°C were used for all analytical work. A detailed linearity and reproducibility study of this colorimetric reaction was completed (Table 2). It was determined that the detection limit was 5 ppm erythromycin at the 592 nm wavelength. At 10 ppm the average absorbance was 0.025 with a relative standard deviation (RSD) of 16%; at 100 ppm the average absorbance was 0.369 with a RSD of 1.7%; at 2 5 0 p p m the average absorbance was 0.938 with a RSD o f 1%. The RSD of the slope at 592 nm was 1%. The detection limit was deter-
3.3. Real sample analysis Because of the better selectivity at 592 nm, all real samples were analyzed at this wavelength despite the sacrifice in sensitivity. The real life samples chosen to be tested were spiked fermentation broths and an erythromycin oral suspension (Table 3). The determination of erythromycin in the oral suspension, a white sample matrix containing suspended solids, gave mixed results. It is believed that the error fluctuation was due to the difficulty in measuring the 1 ml aliquot of the oral suspension or the inhomogeneity of the sample. More work is necessary to decide if pharmaceutical samples can be assayed in this way. The fermentation broths represented a more challenging matrix due to their dark brown color as well as suspended solids. The assay of fermentation broths is commercially important in order to determine which microorganisms are effective in producing antibiotics in high yield. The expected range for macrolide antibiotics in fermentation broths is from about 1 to 10ppt (mgml-~). Therefore,
Table I Effect of water in the ferric ion solution on the absorbance of the Fe a+ - e r y t h r o m y c i n product at 592 n m A m o u n t o f water added to 100ml o f ferric chloride solution (ml)
Average absorbance (n = 3)
Standard deviation
Relative standard deviation
5 2 I 0.5
0.317 1.141 0.741 0.750
0.00289 0.00785 0.0197 0.0497
0.00912 0.00688 0.0265 0.0663
Table 2 Spectrophotometric linearity of the erythromycin-ferric ion complex Wavelength (nm)
Data points
Linear range (mgl -t)
Least squares u
Correlation coefficient
592
29
5-250
0.9997
484
16
2.5-250
m=3.9xi0 -~_+3.9xl0 -s b = - 9 . 2 x 10 -~ + 5.1 x 10 --~ m = 5.92 x 10 -3 + 1.5 x 10 - 4 b = 2.90 x 10 - 2 + I.g 4- 10 - 2
Equations
Y = m x + b.
0.9979
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432 =
4, Conclusion
4000
, ~ ,
3000
lie
~
e
"
e, f
2000"
a~
,.: ,.,. l : ; ; . . . . . . ~ * " * " ': :."o la D o :: iio m , Room Temperature • 40Oc
1000"
g*:
<:
• 50Oc
• 60oc r . . . . . . . . . . . .
0 :E
1431
0
10
ZO
30
40
Time
train)
50
60
70
Fig. 8. Optimization of the molar absorptivity as a function of temperature for the Fe~+-hemiketal reaction.
three samples of Difco broth were spiked with erythromycin from 0.8-5 ppt. The recoveries at 2 and 5 ppt averaged 103% while that at 0.8 ppt was 94%. The lower recovery at 0.8ppt may be because the actual erythromycin determined was at the low end of the linear range, 25 ~tg ml-~. The recovery of tylosin at 10 ppt from the Eli Lilly broth was good. The results from these spiked samples imply that this technique could easily and accurately determine the macrolide antibiotic concentration in actual incubating fermentation broths.
It was determined that erythromycin will react with ferric ion in the presence of an acetic acid-sulfuric acid mixture to form a colored product likely due to a tetraenylic cation in the macrolide ring. The crythromycin molar absorptivities are about 29001mol-Jcm-~ at 592nm and 44001 mol -~ cm -~ at 484 nm. This colorimetric reaction is general for similar macrolide antibiotics except troleandomycin. Because this method requires two 15-30min heated reaction steps, automation by flow injection would likely be diffcult. Although dilution of aqueous samples in concentrated acetic acid is required, this simple inexpensive colorimetric method can be used on fermentation broth samples with an average recovery of 9 9 % .
Acknowledgments Summer support was provided by an NSF Undergraduate Research grant. We thank A. Tietz of Eli Lily for on of the fermentation broths and T. Pekol for assistance with the LC-MS study.
Table 3 Percent recovery of erythromycin and tylosin from various sample matrices Concentration of antibiotic Sample
Added
Difco fermentation broth
Erythromycin
# 1 #2 #3 # 4 # 5 # 6 #7 #8
Eli Lily fermentation broth
Tylosin
# I
Recovery Found
(%)
2.01 mg m l - ~ 2.01 mg m l - ' 5.01 mg m l - ' 5.01 mg ml - ' 810 lag m l - i 810 lag m l - '
2.07 mg m l - ' 2.19 mg m l - ' 5.12 mg ml -~ 4.96 mg m l - ' 720 lag m l - i 820 lag m l -
810 lag ml -s
760 lag ml - t
810 lag ml - I
760 pg ml - I
103 108 102 99.0 88.9 101 93.8 93.8
10.0 mg ml -~
Oral suspension
Erythromycin
# I #2 # 3 # 4
400 lag ml -
~
9.96 mg ml -~ 424 pg m l - i
400 lag ml - I
417 lagml - I
400 lag ml - i 400 lag m l - i
329 lag m l - i 299 lag m l - '
99.6 106 104 82.3 74.8
1432
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432
References [1] N.D. Danielson, J.A. Holeman, D.C. Bristol and D.H. Kirzner, J. Pharm. Biomed. Anal., I1 (1993) 121. [2] J.H. Ford, G.C. Prescott, J.W. Hinman and E.L. Caron, Anal. Chem., 25 (1953) 1195. [3] V.B. Korchangin, K.I. Surkove, N.V. Stepushkina and N.A. Vokulenko, Antibiotiki, 7 (1962) 962. [4] P.E. Manni and J.E. Sinesheimer, Anal. Chem., 33 (1961) 1900. [5] D. Dabrowska, A. Regosz, L. Tamkun and E. Kaminska, Sci. Pharm., 52 (1984) 220.
[6] D. Dabrowska, A. Regosz, R. Piekos, M. Mierzwa and B. Paruch, Microchem. J., 41 (1990) 210. [7] A.S. Issa, M.A. Abdel Salam, H.M.G. Daabees and N.S. Boni, Alexandria J. Pharm. Sci., 4 (1990) 7. [8] M. Tsuneko, J. Pharm. Soc. Jpn., 79 (1959) 29. [9] F.U Neely, Anal. Chim. Acta, 281 (1993) 243. [10] R.W. Burke, B.I. Diamondstone, R.A. Velpoldi and O. Menis, Clin. Chem., 20 (1974) 794. [11] P. Kurath, P.H. Jones, R.S. Egan and T.J. Perun, Experientia, 27 (1970) 362.
:
•
" -.'i.'"~"
,
r~
:
',"?!
Talanta ELSEVIER
Talanta 42 (1995) 1425-1432
Colorimetric determination of macrolide antibiotics using ferric ion Patricia A. Gallagher, Neil D. Danielson
*
Department o1 Chemistry, Miami Unit,ersity, Oxfbrd, OH 45056, USA
Received 8 December 1994; revised 13 March 1995; accepted 14 March 1995
Abstract
Macrolide antibiotics such as erythromycin, oleandomycin, spiromycin, and tylosin are found to react with Fe 3+ in the presence of an acetic acid-sulfuric acid mixture to form a colored product having a useful absorption band at 592 nm. Troleandomycin forms only a weakly colored product upon reaction. The molar absorptivity is about 29001mol -~ c m - ' for erythromycin and the detection limit is 5 lag ml-~. This colorimetric method permits the analysis of fermentation broths containing either erythromycin or tylosin without a separation step.
1. Introduction
Macrolide antibiotics, all composed of either a 14-membered or a 16-membered oxygenated ring with several sugars attached, have proved to be effective antibacterial agents for m a n y years. The structures of five c o m m o n macrolide antibiotics are shown in Fig. I. Erythromycin is certainly the most well-known compound of this class; however troleandomycin is also available as a pharmaceutical product and tylosin is used widely by veterinarians [1]. At least six colorimetric methods have previously been developed for several of these macrolide antibiotics. Hydrolysis of the sugar groups o f erythromycin [2] or oleandomycin [3] in concentrated sulfuric acid produced a yellow product measurable at 470 nm. This reaction has recently been extended to the other three macrolide antibiotics shown in Fig. l with a comparison o f linearity and detection limits [l]. Tetrazolium blue can react generally with reducing agents such as erythromycin to form a dark blue solution [4]. Complex formation with * Corresponding author.
Bromcresol Purple has been applied to the colorimetric determination of erythromycin [5]. A variety of other dyes which undergo ion pair formation with erythromycin in chlorinated solvents can be utilized [6]. Erythromycin and tylosin have been determined at 842 nm using tetracyanoquinodimethane [7]. A coiorimetric method at 532 nm for oleandomycin using diazotized sulphanilic acid is known [8]. However, all of these methods are nonspecific or require a nonaqueous solvent and would be difficult to use for the determination o f macrolide antibiotics in aqueous complicated matrices such as fermentation broths without a prior separation step. The reaction of cis-aconitic anhydride with a tertiary amine such as a macrolide antibiotic permits the formation of a colored product detectable at 525 nm. Although this reaction has been applied to the determination o f tylosin in fermentation broths, extraction of the broth with sufficient isopropyl acetate to give a residue that could be reconstituted to attain a ! m M concentration level is required [9]. We report here a coiorimetric method for macrolide antibiotics which involves the
0039-9140/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0039-9140(95)01587-6
1426
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-/432 o
,,,=,-" "o. -'-F -o~..~-=,-,=,
ERYTHROMYCIN A
=1
Ih
Ih
m=
o~,
n
o
,,~"~?-"'r" "'o3.~g=,,
OLEANDOMYCIN TROLEANDOMYCIN
./
SPIRAMYCIN
~'~
=,
=-u =--¢o0~
~=~
.%=,
== I
O
TYLOSIN Fig. 1. Structures of macrolide antibiotics.
macrolide ring and not the sugar or tertiary nitrogen moiety. After dehydration of the macrolide antibiotic in concentrated acetic acid, the addition of Fe 3+ will generate a coloured product measurable at 592 nm. The determination of erythromycin or tylosin in fermentation broths is possible without sample pretreatment.
2. Experimental 2.1. Chemicals
The macrolide antibiotics (Fig. 1) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The erythromycin ethylsuccinate and sulfisoxazole acetyl oral suspension was
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432
obtained from Abbott Laboratories (North Chicago, IL, USA). There were two different fermentation broths used for this research. One was a dehydrated Antibiotic Medium 3 purchased from Difco Laboratories (Detroit, MI, USA) and the second, donated by Eli Lilly (Indianapolis, IN, USA) was a dehydrated fermentation broth used for tylosin production. The Difco product was composed of beef extract, yeast extract, peptone, dextrose, sodium chloride, dipotassium phosphate, and monopotassium phosphate. The Eli Lilly sample was made up of yellow corn meal, fish meal, corn gluten meal, sodium chloride, ammonium phosphate, calcium carbonate, crude soybean oil, and cane molasses. The ferric chloride was purchased from MCB Manufacturing Chemists, Inc. (Cincinnati, OH, USA). The sulfuric acid and acetic acid were both purchased from Fisher Scientific (Cincinnati, OH, USA) and the triply distilled water obtained from a Barnstead Nanopure distillation unit (Sybron/Barnstead, Dubuque, IA, USA). 2.2. Equipment The spectrophotometry was conducted either on a Varian (Humboldt, CA, USA) DMS 90 UV-ViS spectrophotometer or a Hewlett-Packard (Palo Alto, CA, USA) model 8452A diodearray instrument. The sample compartment was heated to 50°C using a Fisher Scientific (Cincinnati, OH, USA) model 900 isotemp refrigerated circulator water bath. 2.3. Procedure A 500 lag ml -j erythromycin stock solution prepared in glacial acetic acid was allowed to react at 45"C for at least 30 min (often 2h) before use. A ferric ion stock solution was prepared by weighing 0.080 g of ferric chloride, pipeting 2 ml concentrated sulfuric acid and 2 ml of triply distilled water into the 100 ml volumetric flask, and diluting to the mark with concentrated glacial acetic acid. Erythromycin-ferric ion standards generally ranging from 10-250 ug ml- ~ were prepared by pipeting 5 ml of ferric ion stock solution and the appropriate amount of erythromycin stock solution before dilution to a 10 ml volume with glacial acetic acid. The fermentation broths were prepared by adding either 17.5 g of dehydrated Antibiotic Medium 3 or the entire Eli Lilly sample to a 1000 ml volumetric flask and
1427
filling to the mark with triply distilled water. A 5 ml aliquot of the spiked fermentation broth sample was placed in a 50 ml volumetric flask and filled to the mark with concentrated glacial acetic acid. The blank was prepared in exactly the same way as the sample except that the fermentation broth was not spiked with the macrolide antibiotic. All fermentation samples and blanks were heated in the 45°C water bath. The standards for this part were prepared exactly the same as for the sample except triply distilled water was used instead of the fermentation broth. The pharmaceutical sample was prepared by measuring out in a volumetric test tube 1 ml of the erythromycin ethylsuccinatesulfisoxazole acetyl oral suspension and quantitatively transfering it to a 100 ml volumetric flask before filling to the mark with concentrated glacial acetic acid. This was heated in the 45"C water bath. The above procedure for preparing the erythromycin-ferric ion standards was carried out for all the samples. All standards, samples and blanks, after mixing with ferric ion, were allowed to react in the heated (50"C) curvette holder for 15 min before the absorbance measurement was taken.
3. Results and discussion
3.1. Proposed reaction mechanism Previously, it had been proposed [10] that the reaction of cholesterol in an acetic acidsulfuric acid mixture with Fe 3÷ will lead to the formation of trienylic and tetraenylic cations detectable at about 480 nm and 560 nm, respectively (Fig. 2). The wavelength maxima are dependent on the sulfuric acid concentration in the Fe 3+ stock solution. It is also known [11] that erythromycin in the presence of concentrated acetic acid can undergo dehydration, forming the 8,9-anhydroerythromycin-6,9hemiketal (Fig. 3). The hydroxy group beta to the double bond in this hemiketal product is a remarkably similar key functional group to that in cholesterol. Using LC-MS, we confirmed the formation of this hemiketal product as evidenced by the disappearance of the M+,MH ÷ ion peak pair at M/z = 734, 735 and the formation of the ion peak at M/z = 716. No peak for the diene carbonium ion was evident. A peak at M/z = 558 present in both the erythromycin and hemiketal spectra represents the fragment ion of erythromycin minus a
1428
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432
~L
HOAc/H2S04
~L
Fe3+
~
'll" Fe2+
3.2. Optimization of colorimetric response
~tion
Fig. 2. Proposed mechanism of the eholesterol-Fe 3÷ reaction (from Ref. [10]).
sugar group. An absorption spectrum of the hemiketal product after reaction with Fe3+using the optimum conditions shows two strong peaks at 484 and 592 nm (Fig. 4). A spectrum of the cholesterol reaction run using an 80% acetic acid-20% sulfuric acid Fe 3+ solution showed three peaks with wavelength
N(CH3)2
H3'~
I~Cl-t3,0...L.~m/~.~
./~-o43 ~,o,.,~% "sC~ b v , k . ~
maxima at 620, 453, and 412 nm. A spectrum of the hemiketal product run using the same 80% acetic acid-20% sulfuric acid Fe 3+ solution also showed three peaks with wavelength maxima of 596, 486, and 412 nm. A glucose stock solution gave no reaction with Fe 3+, providing furthur evidence that the likely source of the colored product was from the macrolide ring and not the sugar groups attached to the macrolide ring. Although L C MS could not provide a clear mass spectrum for the formation of trienylic or tetraenylic cations for the Fe3+-erthyromycin reaction, we feel this is the likely type of colored product.
,,_L~oN
H3C 0(343 HOAc N(C83)2
•
Fig. 3. Acetic acid degradation of erythromycin A to form the hemiketal product.
A time study of the dehydration reaction in concentrated acetic acid at 45°C for all five compounds shown in Fig. 1 was carried out (Fig. 5). All the macrolide antibiotics except troleandomycin seemed to undergo significant dehydration to form a product that would react with Fe 3+ ion. Because troleandomycin has an ester group instead of the hydroxyl group as in oleandomycin, this difference in reactivity is not surprising. Both spiramycin and tylosin already have some conjugation in their structures which is apparently expanded after reaction with Fe 3+. The spectra of the Fe3+-antibiotic products for oleandomycin, spiromycin, and tylosin were similar to that of erythromycin. The following optimum heating times for the macrolide antibiotic stock solutions were 120 min for erythromycin, 90 min for oleanomycin, 60 min for spiromycin and 0 min for tylosin. Although the optimum heating times were quite varied, in general a reaction time of only 30 rain would be sufficient for good dehydration to the Fe 3+ reactive product. The effect of water in the erythromycin stock solution on the colorimetric reaction was studied (Fig. 6). It appears that 20% water can lower the absorbance to zero; only 5% water in the acetic acid solution caused a 50% decrease in the absorbance of the Fe3+-hemiketal product. Therefore, it was decided that for best detectability the macrolide antibiotic stock solution in acetic acid should be a prepared if possible from a solid instead of an aqueous sample. The proper amount of sulfuric acid needed to be added to the ferric chloride stock solution prepared in glacial acetic acid to give maxi-
1429
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432 1.5001
1.2193
J
0.9385 4C m
$ us m ,i[
0.8577
0.3769
O.Og60
.
400
.
.
.
.
. . 500
.
.
.
. 600
1 700
. . . .
~0
"
Wavelength (am) Fig. 4. Absorbance spectrum of the Fe3+-hemiketal reaction product of erythromycin (250 pg ml-t).
mum absorbance was determined. Ferric chloride solutions containing 1, 2 and 4 ml o f sulfuric acid were prepared and time studies were completed (Fig. 7). In comparison, the cholesterol-Fe 3+ reaction would not work using such low concentrations o f sulfuric acid. The absorbance profiles for the 2 and 4 ml solutions were almost exactly the same. There were some reproducibility problems; the inconsistency in the results were believed to be caused by the sulfuric acid absorbing water. To alleviate this problem, small amounts o f water were added to the ferric chloride stock
solution (Table 1). Therefore, it was determined that 2 ml o f water as well as 2 ml o f sulfuric acid should be added to 100ml o f ferric chloride solution to obtain the optimum absorbance and reproducibility. At room temperature, the F e 3 + - a n h y d r o hemiketal macrolide reaction rate is very slow; therefore, the solution was heated at 40, 50 and 60°C (Fig. 8). At 50°C, the data obtained produced a reproducible plateau from approximately 10 to 30 min. Some loss in signal was observed after 10rain using a 60°C reaction temperature. Therefore reaction con-
OAt 1.2 m
et
•
0.S
o
1.o n
•
m
6
.
m
i
0.4
0.8'
•~
0.I'
fl
<
O. 4
"
[ • Ery~
I " T'."
II
(3.414N I 10414)
l
I • TmlealldOiyCia (3.0"71 • IO"+bl}J • • .+ I • Slmmmymn (2.903 • 10 M ) 1
O.,"
O.0'
•
•
(~L729 x IO'ald ,
I
•
"~
0.3-
~
0.|"
m
8
0.1
II
O.O
o T i m e Sample Stock Solution w a s betted (rain)
Fig. 5. Reaction of macrolide antibiotics with acetic acid at 45°C as a function of time. The reaction was followedat 592 nm using Fe~+.
,o mL of water per
~o 100 mL of Ery~'omycin
3o Solution
Fig. 6. Effect of water in the erythromyein stock solution on the absorbance. The reaction was followed at 592 nm using Fe "~÷.
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432
1430
mined to be 2.5 ppm of erythromycin at a wavelength of 484 nm; however, the linearity at 484 nm was not as good as at the 592 nm. The RSD of the slope at 484 nm was 2.5%.
3000"
I I I I I V h Ill l I O N I ~ ' ~ , h
-_=
•m••a
2000" >,
1000'
a I
~ ,a
<
~ u aaD
/ n R.T., lmL acid [ • R.T., 2mL acid ] [ • R.T., 4mLacidl
0
o
20
40
60
80
i 100
Time (rain) Fig. 7, Optimization of the molar absorptivity as a function of sulfuric acid volume in the Fe 3+-acetic acid solution. The reaction was followed at room temperature (RT) at 592 n m using Fe 3+.
ditions of 15 min at 50°C were used for all analytical work. A detailed linearity and reproducibility study of this colorimetric reaction was completed (Table 2). It was determined that the detection limit was 5 ppm erythromycin at the 592 nm wavelength. At 10 ppm the average absorbance was 0.025 with a relative standard deviation (RSD) of 16%; at 100 ppm the average absorbance was 0.369 with a RSD of 1.7%; at 2 5 0 p p m the average absorbance was 0.938 with a RSD o f 1%. The RSD of the slope at 592 nm was 1%. The detection limit was deter-
3.3. Real sample analysis Because of the better selectivity at 592 nm, all real samples were analyzed at this wavelength despite the sacrifice in sensitivity. The real life samples chosen to be tested were spiked fermentation broths and an erythromycin oral suspension (Table 3). The determination of erythromycin in the oral suspension, a white sample matrix containing suspended solids, gave mixed results. It is believed that the error fluctuation was due to the difficulty in measuring the 1 ml aliquot of the oral suspension or the inhomogeneity of the sample. More work is necessary to decide if pharmaceutical samples can be assayed in this way. The fermentation broths represented a more challenging matrix due to their dark brown color as well as suspended solids. The assay of fermentation broths is commercially important in order to determine which microorganisms are effective in producing antibiotics in high yield. The expected range for macrolide antibiotics in fermentation broths is from about 1 to 10ppt (mgml-~). Therefore,
Table I Effect of water in the ferric ion solution on the absorbance of the Fe a+ - e r y t h r o m y c i n product at 592 n m A m o u n t o f water added to 100ml o f ferric chloride solution (ml)
Average absorbance (n = 3)
Standard deviation
Relative standard deviation
5 2 I 0.5
0.317 1.141 0.741 0.750
0.00289 0.00785 0.0197 0.0497
0.00912 0.00688 0.0265 0.0663
Table 2 Spectrophotometric linearity of the erythromycin-ferric ion complex Wavelength (nm)
Data points
Linear range (mgl -t)
Least squares u
Correlation coefficient
592
29
5-250
0.9997
484
16
2.5-250
m=3.9xi0 -~_+3.9xl0 -s b = - 9 . 2 x 10 -~ + 5.1 x 10 --~ m = 5.92 x 10 -3 + 1.5 x 10 - 4 b = 2.90 x 10 - 2 + I.g 4- 10 - 2
Equations
Y = m x + b.
0.9979
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432 =
4, Conclusion
4000
, ~ ,
3000
lie
~
e
"
e, f
2000"
a~
,.: ,.,. l : ; ; . . . . . . ~ * " * " ': :."o la D o :: iio m , Room Temperature • 40Oc
1000"
g*:
<:
• 50Oc
• 60oc r . . . . . . . . . . . .
0 :E
1431
0
10
ZO
30
40
Time
train)
50
60
70
Fig. 8. Optimization of the molar absorptivity as a function of temperature for the Fe~+-hemiketal reaction.
three samples of Difco broth were spiked with erythromycin from 0.8-5 ppt. The recoveries at 2 and 5 ppt averaged 103% while that at 0.8 ppt was 94%. The lower recovery at 0.8ppt may be because the actual erythromycin determined was at the low end of the linear range, 25 ~tg ml-~. The recovery of tylosin at 10 ppt from the Eli Lilly broth was good. The results from these spiked samples imply that this technique could easily and accurately determine the macrolide antibiotic concentration in actual incubating fermentation broths.
It was determined that erythromycin will react with ferric ion in the presence of an acetic acid-sulfuric acid mixture to form a colored product likely due to a tetraenylic cation in the macrolide ring. The crythromycin molar absorptivities are about 29001mol-Jcm-~ at 592nm and 44001 mol -~ cm -~ at 484 nm. This colorimetric reaction is general for similar macrolide antibiotics except troleandomycin. Because this method requires two 15-30min heated reaction steps, automation by flow injection would likely be diffcult. Although dilution of aqueous samples in concentrated acetic acid is required, this simple inexpensive colorimetric method can be used on fermentation broth samples with an average recovery of 9 9 % .
Acknowledgments Summer support was provided by an NSF Undergraduate Research grant. We thank A. Tietz of Eli Lily for on of the fermentation broths and T. Pekol for assistance with the LC-MS study.
Table 3 Percent recovery of erythromycin and tylosin from various sample matrices Concentration of antibiotic Sample
Added
Difco fermentation broth
Erythromycin
# 1 #2 #3 # 4 # 5 # 6 #7 #8
Eli Lily fermentation broth
Tylosin
# I
Recovery Found
(%)
2.01 mg m l - ~ 2.01 mg m l - ' 5.01 mg m l - ' 5.01 mg ml - ' 810 lag m l - i 810 lag m l - '
2.07 mg m l - ' 2.19 mg m l - ' 5.12 mg ml -~ 4.96 mg m l - ' 720 lag m l - i 820 lag m l -
810 lag ml -s
760 lag ml - t
810 lag ml - I
760 pg ml - I
103 108 102 99.0 88.9 101 93.8 93.8
10.0 mg ml -~
Oral suspension
Erythromycin
# I #2 # 3 # 4
400 lag ml -
~
9.96 mg ml -~ 424 pg m l - i
400 lag ml - I
417 lagml - I
400 lag ml - i 400 lag m l - i
329 lag m l - i 299 lag m l - '
99.6 106 104 82.3 74.8
1432
P.A. Gallagher, N.D. Danielson / Talanta 42 (1995) 1425-1432
References [1] N.D. Danielson, J.A. Holeman, D.C. Bristol and D.H. Kirzner, J. Pharm. Biomed. Anal., I1 (1993) 121. [2] J.H. Ford, G.C. Prescott, J.W. Hinman and E.L. Caron, Anal. Chem., 25 (1953) 1195. [3] V.B. Korchangin, K.I. Surkove, N.V. Stepushkina and N.A. Vokulenko, Antibiotiki, 7 (1962) 962. [4] P.E. Manni and J.E. Sinesheimer, Anal. Chem., 33 (1961) 1900. [5] D. Dabrowska, A. Regosz, L. Tamkun and E. Kaminska, Sci. Pharm., 52 (1984) 220.
[6] D. Dabrowska, A. Regosz, R. Piekos, M. Mierzwa and B. Paruch, Microchem. J., 41 (1990) 210. [7] A.S. Issa, M.A. Abdel Salam, H.M.G. Daabees and N.S. Boni, Alexandria J. Pharm. Sci., 4 (1990) 7. [8] M. Tsuneko, J. Pharm. Soc. Jpn., 79 (1959) 29. [9] F.U Neely, Anal. Chim. Acta, 281 (1993) 243. [10] R.W. Burke, B.I. Diamondstone, R.A. Velpoldi and O. Menis, Clin. Chem., 20 (1974) 794. [11] P. Kurath, P.H. Jones, R.S. Egan and T.J. Perun, Experientia, 27 (1970) 362.