The Stability of Calcium Glucoheptonate Solutions
THE STABILITY OF CALCIUM GLUCOHEPTONATE SOLUTIONS
BY
RAJAGOPALAN SURYANARAYANAN
M. Pharm., Banaras Hindu University, 1978
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
THE FACULTY OF GRADUATE STUDIES
(Faculty of Pharmaceutical Sciences)
Division of Pharmaceutics
We accept this thesis as conforming
to the required standard
c RAJAGOPALAN SURYANARAYANAN, 1981 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of
The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5
Date - ii -
ABSTRACT
Calcium glucoheptonate is official in the USP XX as Calcium
Gluceptate and is described as the calcium salt of D-glycero-D-gulo-
heptonic acid which is the a epimer of glucoheptonic acid. It is
freely soluble in water. Since late 1976, solutions of calcium gluco•
heptonate have shown a tendency to precipitate on storage. The problem
of precipitation can be due to one or more of the following reasons:
(i) change from an unstable to a stable modification
(ii) presence of seed crystals
(iii) differing proportions of a and 3 epimers in the calcium
glucoheptonate obtained from various sources
Calcium glucoheptonate was found to be amorphous while the precipi•
tate was crystalline. Membrane filtration increased the time taken
for precipitation to occur while autoclaving resulted in stable solutions.
It can be postulated that the majority of seed crystals are excluded by
filtration which results in increased stability and autoclaving destroys
the seed crystals.
When calcium glucoheptonate from different sources was used for
the preparation of solutions, the time for precipitation varied with
the commercial source (Table 1). The most stable solution was prepared from a salt described as calcium a-B glucoheptonate and the least stable was supplied as Calcium Gluceptate USP. - iii -
Table 1. Stability of calcium glucoheptonate solution 26.7% w/v
and proportion of a epimer in various commercial samples.
Time for precipita• Proportion of a epimer Source tion (days) (percent)
Pfanstiehl a-8' stable 51.8
Givaudan 8 71 .8
Italsintex 2 72.4
Pfanstiehl USP <1 100
It therefore seemed likely that, in addition to the presence of seed crystals, stability may depend on the relative proportions of the a and 3 epimers. It is interesting that to comply with the USP specifications, calcium glucoheptonate should be the unstable a epimer, although no procedure is given in the monograph for the identification or assay of the a form. Hence methods have been developed to identify and to deter• mine the proportions of the a and 6 epimers.
An aqueous solution of calcium glucoheptonate was converted into a mixture of glucoheptonic acids and their corresponding y lactones by passage through a cation exchange column. The solution was freeze-dried and the acid-lactone mixture was completely converted to the y lactones using concentrated HCl.
Trimethylsilyl (TMS) derivatives of the lactones were formed by reaction with trimethylsilylimidazole in pyridine. Gas chromatography on a 3% OV-225 column using a flame ionization detector gave two peaks. - iv -
A control experiment using the TMS derivative of a reference sample
of the y lactone of a-D-glucoheptonic acid gave a single peak having the
same retention time as the second peak of the sample, thereby indicating
that the second peak is due to the TMS derivative of this y lactone.
The two GC peaks gave similar mass spectral patterns and subjecting the
reference material to the same GC-MS analysis, confirmed that the peak
2 was the TMS derivative of the y lactone of a-p-glucoheptontecacid. Since
the GC peaks 1 and 2 have different retention times but the same molecular
ion and similar fragmentation patterns, the chemical structures of the
two compounds must be very similar and hence peak 1 is attributed to the
TMS derivative of the y lactone of 3-D-glticohe.ptontc acid. The relative
proportions of the a and 6 epimers were calculated using the TMS deriva•
tive of sucrose as an internal standard. The correlation between the
stability results and the proportions of the a and 6 epimers in various
commercial samples of calcium glucoheptonate is shown in Table 1.
Hence it seems that all the three reasons postulated earlier have
some role to play in the recrystallization of calcium glucoheptonate.
By the use of elemental analysis, IR spectroscopy, DSC and GC-MS
studies, the precipitate obtained from solutions of calcium glucoheptonate
has been identified as a hydrate of calcium glucoheptonate.
Attempts were made to develop stable oral and parenteral solutions
containing glucoheptonate. All the oral formulations commenced precipita•
tion within six months. Stable parenteral formulations can be prepared by autoclaving the final solution. If sterilization by filtration is desired, then the solution can be stabilized with calcium D-saccharate or calcium gluconate. - V -
TABLE OF CONTENTS
Pa^e
ABSTRACT i i
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF SCHEMES xii
LIST OF ABBREVIATIONS xiii
ACKNOWLEDGEMENTS xiv
PART A - THE STABILITY OF CALCIUM GLUCOHEPTONATE SOLUTIONS
1. INTRODUCTION 1
1.1 METHODS OF ANALYSIS OF CALCIUM GLUCOHEPTONATE 3
1.2 CHROMATOGRAPHY OF HEPTOSES AND HEPTONOLACTONES ' 3
1.2.1 Gas chromatography 3
A. Methyl derivative 4
B. Trimethylsilyl derivative 8
1.2.2 Paper chromatography 9
1.3 LACTONIZATION OF ALDONIC ACID 9
1.4 MASS SPECTROMETRY OF HEPTONOLACTONES 11
1.5 PHASE TRANSITIONS 11
1.5.1 Amorphous-crystalline transitions . 12
1.5.2 Hydrate anhydrous form transitions 13
1.5.3 Polymorphic transitions 14 - vi -
Page
2. EXPERIMENTAL 15
2.1 APPARATUS 15
2.2 MATERIALS 16
2.3 STABILITY STUDIES OF CALCIUM GLUCOHEPTONATE SOLUTIONS 18
2.3.1 Solutions made from calcium glucoheptonate after heating at 120°C 18
2.3.2 Heat treatment of calcium glucoheptonate solutions 19
2.3.3 Membrane filtration of calcium glucoheptonate solutions 19
2.4 CHARACTERIZATION OF CALCIUM GLUCOHEPTONATE AND THE PRECIPITATE OBTAINED FROM SOLUTIONS OF CALCIUM GLUCOHEPTONATE 19
2.4.1 Elemental analysis 19
2.4.2 Thermal analysis 20
2.4.3 Infrared spectra 20
A. Preparation of solid samples 20
B. Preparation of solutions 20
2.4.4 Heat of solution 21
2.4.5 X-ray diffraction * 21
2.4.6 Equilibrium solubility 21
2.5 DEVELOPMENT OF A GAS CHROMATOGRAPHIC METHOD FOR ESTIMATING THE PROPORTIONS OF a AND 3 EPIMERS IN CALCIUM GLUCO• HEPTONATE 21
2.5.1 Selection of a substance for preliminary studies 21
2.5.2 Preparation of methyl derivative 22
A. Preparation of sodium methylsulfinylmethide 22
B. Derivative formation 22 - vii -
Page
2.5.3 Preparation of trimethylsilyl derivative 23
A. N-trimethylsilylimidazole 23
B. Mixture of trimethylsilylimidazole, N,0-bis(trimethylsilyl) acetamide, and trimethylchlorosilane 23
C. Trimethylsilylimidazole in pyridine 24
2.5.4 Preparation of the trimethylsilyl derivative
of calcium glucoheptonate 24
2.5.5 Lactone formation 27
2.5.6 Gas chromatography-mass spectrometry .30
2.5.7 Selection of internal standard 32
2.5.8 Optimization of GC conditions 32
A. Selection of stationary phase 33
B. Temperature programming 33
C. Injection temperature 34
D. Detector temperature 34
E. Optimization of reaction time 34
2.6 PREPARATION OF STANDARD CURVE OF THE y-LACTONE OF a-D-GLUCOHEPTONIC ACID 36
2.7 DETERMINATION OF THE PROPORTIONS OF a AND 3 EPIMERS IN COMMERCIAL SAMPLES OF CALCIUM GLUCOHEPTONATE 36
2.8 DETERMINATION OF THE PROPORTIONS OF a AND 3 EPIMERS IN THE PRECIPITATE OBTAINED FROM COMMERCIAL SAMPLES OF CALCIUM GLUCOHEPTONATE 38
3. RESULTS AND DISCUSSION 39
3.1 STABILITY STUDIES OF CALCIUM GLUCOHEPTONATE SOLUTIONS 39
3.2 CHARACTERIZATION OF CALCIUM GLUCOHEPTONATE AND THE PRECIPITATE OBTAINED FROM SOLUTIONS OF CALCIUM GLUCOHEPTONATE 39 - viii -
Page
3.2.1 Elemental analysis 39
3.2.2 Thermal analysis 39
3.2.3 IR spectra 42
A. Solid samples 42
B. Solutions 47
3.2.4 Heat of solution 47
3.2.5 X-ray diffraction studies 47
3.2.6 Equilibrium solubility 53
A. Calcium glucoheptonate 53
B. Precipitate dried under vacuum at room temperature to constant weight 53
C. Precipitate dried under vacuum at 80°C for
46 hours 54
3.3 IDENTIFICATION OF THE PRECIPITATE 54
3.4 POSSIBLE REASONS FOR PRECIPITATION 55
3.4.1 Change from an unstable form to a stable form 55
3.4.2 Presence of seed crystals inducing crystallization 56
3.4.3 Differing proportions of the a and p! epimers in the calcium glucoheptonate from various sources 57 3.4.4 Some comments about USP specifications of calcium glucoheptonate 60
PART B - DEVELOPMENT OF ORAL AND PARENTERAL LIQUID DOSAGE
FORMS CONTAINING CALCIUM GLUCOHEPTONATE
1. INTRODUCTION . 63
2. EXPERIMENTAL 64
2.1 MATERIALS 64 - ix -
Page
2.2 DEVELOPMENT OF ORAL FORMULATIONS 65
2.2.1 Basic formula 65
2.2.2 Use of sugar 65
2.2.3 Use of stabilizing agent 65
2.2.4 Method of preparation of oral formulations 68
2.3 DEVELOPMENT OF PARENTERAL FORMULATIONS 70
2.3.1 Basic formula 70
2.3.2 Use of stabilizing agents 70
2.3.3 Method of preparation of parenteral formulations 70
3. RESULTS AND DISCUSSION
3.1 STABILITY STUDIES OF ORAL FORMULATIONS 71
3.2 STABILITY STUDIES OF PARENTERAL FORMULATIONS 71
3.2.1 Sterilization by autoclaving 71
3.2.2 Sterilization by filtration 71
SUMMARY 76
REFERENCES 78 - X -
LIST OF TABLES
Table Page
I . Stability of calcium glucoheptonate 40 (26.7% w/v) in aqueous solution
II Elemental composition of calcium gluco- 41 heptonate and the precipitate obtained from the solution of calcium glucoheptonate
III Heats of solution of calcium glucoheptonate 48 samples
IV X-ray studies of the precipitate obtained 49 from the solution of calcium glucoheptonate USP (Pfanstiehl)
V Determination of the proportion of the 58 a epimer in various commercial samples of calcium glucoheptonate
VI Relationship between the proportion of 59 the a epimer and the stability of calcium glucoheptonate in solution
VII Proportion of the a epimer in the precipitate 61 obtained from solutions of calcium gluco• heptonate
VIII Basic formula 66
IX Modified basic formula containing sugar as 67 sweetening agent
X Oral calcium syrup-formulation details 69
XI Oral calcium syrup-stability studies 72 (calcium glucoheptonate USP (Pfanstiehl)
XII Oral calcium syrup-stability studies 73 (calcium glucoheptonate - Givaudan)
XIII Calcium injection-stability studies 74 (calcium glucoheptonate USP - Pfanstiehl) - xi -
LIST OF FIGURES
Figure Page
1 Structure of calcium salts of a and $-D- 2 glucoheptonic acid
2 Structure of a and B-D-glucoheptose 5
3 Structure of y and 6-lactones of a-D- 6 glucoheptonic'acid
4 Structure of y and 6-lactones of 3-D- 7 glucoheptonic acid
5 Chromatogram of the TMS derivatives of the 28 y-lactones of a and 3-D-glucoheptonic acid
6 Chromatogram of the TMS derivative of the 29 y-lactone of a-D-glucoheptonic acid (reference material)
7 Chromatogram of the TMS derivative of the 35 Y-lactones of a and 3-D-glucoheptonic acid. with the TMS derivative of sucrose as internal standard
7a Standard curve of the y-lactone of a-D- 37 glucoheptonic acid
8 Infrared spectra of calcium glucoheptonate 43 and the precipitate (KBr disc)
9 Infrared spectra of calcium glucoheptonate 45 and the precipitate (solution in chloroform) - xn -
LIST OF SCHEMES
Lactonization of a and B-D-glucoheptonic acid
Fragmentation scheme of the TMS derivatives of the y-lactones of a-D-glucoheptonic acid and B-D-glucoheptonic acid - xiii -
LIST OF ABBREVIATIONS
DSC differential scanning calorimetry
GC gas chromatography
GC-MS gas chromatography-mass spectrometry
IR infrared
RT room temperature
TMS trimethylsilyl
USP United States Pharmacopoeia ACKNOWLEDGEMENTS
I am thankful to the following people for their help during the course of this work:
Dr. A.G. Mitchell, Dr. H.M. Burt, Dr. F.S. Abbott,
Dr. K.M. McErlane, Dr. J.H. McNeill, Mr. R. Butters,
Dr. J.N.C. Whyte, Mr. R. Burton, Mr. R. Goring,
Pillai, Marvin, Sheila and Andrew.
The financial assistance provided by the Science Council of
British Columbia and Stanley Drug Products Ltd. is gratefully acknowledged.
Thanks to Stanley Drug Products Ltd. for their generous gift of a number of chemicals. - XV -
To
Amma & Babuji - xvi -
PART A
THE STABILITY OF CALCIUM GLUCOHEPTONATE SOLUTIONS - 1 -
1. INTRODUCTION
Calcium glucoheptonate is used in the treatment of calcium deficiency
(Wade, 1977a). According to the United States Pharmacopoeia (USP ;XX,1980)
it is anhydrous or it contains varying amounts of water of hydration.
The a epimer of calcium glucoheptonate, also known as calcium D-gTycero-
D-gulo heptonate is official in the USP. However, some of the commercially
available calcium glucoheptonate appears to be a mixture of the a and 3
epimers of calcium glucoheptonate.. The 3 epimer is chemically known as
calcium-D-glycero-D-ido heptonate. These two forms differ only in their
configuration about the C-2 carbon atom (Fig. 1).
Calcium glucoheptonate is a unique product in that it has a very
high aqueous solubility. Solutions of calcium glucoheptonate containing
85 percent solids have been prepared which have not crystallized on
prolonged standing (Product manual, Pfanstiehl). However, since late
1976, a tendency to crystallize on storage has been reported (Muller and
others, 1979; Chiu and Goring, 1979; Holstein, 1980).
According to Muller and others (1979) the precipitate obtained from a solution of calcium glucoheptonate is capable of existing in two forms -
Form A and Form B. The presence of seed crystals of Form A was thought
to be responsible for the precipitation. Heating calcium glucoheptonate powder to 115-120°Cwas said to destroy the Form A crystals, but this method offered no absolute guarantee of stability. Heating solutions of calcium glucoheptonate to a minimum temperature of 80^ for 30 minutes, was said to destroy the Form A crystals and offer complete protection against later crystallization. Chou and Goring (1979) suggest that a - 2 -
COO - rcoo - 1 2 H - C OH OH - c - H 1 . 1 H - C OH H - 3c - OH Ca Ca . I 1 H OH - c OH - \ - H 1 H - c OH H - 5c - OH 1 H OH H - \ - OH I1
CH2OH 7 CH2OH
calcium a-D-glucoheptonate calcium 3-D-glucoheptonate calcium D-glycero-D-gulo-heptonate calcium D-glycero-D-ido-heptonate
Fig. 1. Structure of calcium salts of a and
g-D-glucoheptonic acid - 3 -
change in the manufacturing process of calcium glucoheptonate could have introduced or removed impurities which initiate the crystallization process. They heated calcium glucoheptonate at 80°Cfor 30 minutes and used it to prepare formulations but crystallization still occurred on storage.
According to one manufacturer (Holstein, 1980), the manufacture.-, of calcium glucoheptonate in the amorphous form has been impossible in recent years. He attributed the problem of crystallization to the presence of seed crystals or to something else initiating crystallization.
Holstein suggests that if the product is prepared hot or autoclaved after packaging, there is no crystallization problem.
1.1 METHODS OF ANALYSIS OF CALCIUM GLUCOHEPTONATE
The pharmacopoeia! (USP XX, 1980) assay method for calcium gluco• heptonate consists of the complexometric estimation of calcium with ethylenediaminetetraacetic acid. The percentage purity of calcium glucoheptonate is determined on the basis of the amount of calcium present in the material. Thus, this assay method is insensitive to the sugar portion of the molecule. A conductometric method (Nikolic and others, 1973) of assay of calcium glucoheptonate has also been reported.
1.2 CHROMATOGRAPHY OF HEPTOSES AND HEPT.0N0LACT0NES
1.2.1 Gas Chromatography (GC)
Muller and others (1979) reported a GC method for the determination - 4 -
of the proportions of the a and 6 epimers in calcium glucoheptonate as well as in the precipitate obtained on storage of the solutions. Trimethyl- silyl (TMS) derivatives were prepared and the two forms separated on a 5% methylphenyl silicone (OV-17) column, Unfortunately, details of the experimental conditions were not presented. It was not clear whether the TMS derivative of calcium glucoheptonate was prepared or whether the derivatization took place after conversion of calcium glucoheptonate to glucoheptonic acid by passage through ion-exchange resin. The gas chromatography of a non-volatile compound like calcium glucoheptonate even after derivatization would be unlikely. On the other hand, if calcium glucoheptonate was converted to glucoheptonic acid, then the formation of the 1,4 and 1,5 lactones of both a and 3-D-glucoheptonic acids is a possibility about which the authors make no mention. There are no other published reports on the gas chromatography of calcium glucohepto• nate. However, gas chromatography of D-glycero-D-gulo-heptose as well as the lactones of a-D-glucoheptonic acid . and B-D-glucoheptonic acid have been reported (structures in Figs. 2-4) (see below).
A. Methyl derivative
Whyte (1973) studied the chromatographic mobility of a number of permethylated alditols and aldonates on stationary phases of varying polarities. Sodium glucoheptonate was also one of the compounds studied.
The best separation of the aldonates was achieved on trifluoropropyl- methyl silicone (QF-1) phase. - 5 -
CHO CHO I I 1 H - C - OH OH - C - H 1 1 H - C - OH H - C - OH • 1 1 OH - C - H OH - C - H
H I H - C - OH - C - OH | H - C - OH H - C - OH | CH2OH CH2OH
D-glycero-D-gulo-heptose D-glycero-D-ido-heptose
Fig. 2. Structure of a and B-D-glucoheptose - 6 -
| C = 0 0 = C 1 I I H - C - OH H - C - OH 0 I I H - C - OH H - C - OH 0 I I
I c _ H OH - C - H I I H - C - OH H - C 1 I I H - C - OH H - C - OH I I
CHo0H CH20H
1,4-lactone of 1,5-lactone of a-D-glucoheptonic acid a-D-glucoheptonic acid (y-lactone of a-D-glucoheptonic (6-lactone of a-D-glucoheptonic acid) acid)
Fig. 3. Structure of y and 5-lactones of g-D-glucoheptonic
> acid. - 7 -
C = 0 0 = C I I I OH - C - H OH - c - H I 1 H - C - OH H - c - OH I 1 - C - H OH - c - H 1 H - C - OH H 1 I - c 1 H - C - OH 1 H - c - OH
CH2OH CH20H
1,4-lactone of 1,5-lactone of 3-D-glucoheptonic acid 3-D-glucoheptonic acid (y-lactone of 8-D-glucoheptonic (6-lactone of 8-D-glucoheptonic acid) acid)
Fig. 4. Structure of y and 5-lactones of
3-D-glucoheptonic acid. - 8 -
B. Trimethylsilyl (TMS) derivative
The separation and estimation of carbohydrates by gas chromatography of TMS derivative has been described by Sweeley and others (1963). The derivitization was achieved by using hexamethyldisilazane and trimethyl- chlorosilane. Using a methylphenyl silicone (SE-52) column, the two anomers of D-glycero-D-gulo-Heptose were separated. A mixture of twelve aldonic lactones and acids including D-glycero-D-gulo-heptono-y-lactone were separated on a methyl silicone (SE-30) open tubular glass capillary column by Szafranek and others (1974). They determined the cyclic to linear structural ratios of a number of lactones and in case of D-glycero-
D-gulo-heptono-y-lactone it was found to be 97:3. Perry and others (1969) prepared all sixteen possible heptono-1,4-lactones by applying the
Kiliani-Fischer cyanohydrin synthesis to all eight of the possible
D-aldohexoses. Each aldohexose gave rise to the expected two epimeric heptonic acids which after 1actonization and trimethylsilylation, were separated by gas chromatography. The complete separation of all sixteen heptonolactones could not be achieved on a single phase but a neopentyl- glycol sebacate polyester liquid phase column appeared to give the most satisfactory separation. Petersson and others (1967a) studied the separation of various aldono-1,4-lactones in a number of stationary phases and found the most satisfactory separation in a QF-1 column.
Their studies indicate that in a SE-52 column, D-glycero-D-guloheptono- lactone and D-glycero-a-mannoheptonolactone have identical retention times. Morrison and Perry (1966) oxidised a number of aldoses to aldonic acids which after conversion to their 1,4-lactones were trimethylsilylated and analyzed by GC. Two stationary phases were investigated, (i) 10% - 9 -
neopentylglycol sebacate polyester and (ii) polyethylene glycol (Carbowax
20 M) and both phases were found to be only partially satisfactory for separation of the lactones.
1.2.2 Paper chromatography
A number of epimeric heptonolactones have been separated by paper chromatography using a variety of solvent systems (Kjolberg and Veil an,
1966). The best separation of the epimeric mixture of D-glycero-D-gulb heptono-y-lactone and D-glycero-D-ido-heptono-y-lactone was achieved in a ethyl acetate-acetic acid-formic acid-water (18:3:1:4) system.
However, as this system was a very poor solvent, the preparative chromatography was successfully carried out using methyl ethyl ketone- ethanol-water (5:2.1) as eluent.
1.3 LACT0NIZATI0N OF ALDONIC ACID
An aldonic acid in solution establishes equilibrium with its 1,4- and 1,5- lactones (Isbell and Frush, 1963). The equilibrium proportions of the constituents vary with temperature, concentration, solvent, and the characteristics of the particular aldonic acid. Lactone formation is promoted by acidic catalysts and by dehydration with a suitable solvent.
1,5-Lactones are usually formed more rapidly than 1,4-lactones. However, a mixture of acid and lactone can be converted to the 1,4-lactone by dissolving it in a suitable solvent (for example, glacial acetic acid- dioxane) containing a trace of hydrochloric acid and concentrating the solution under reduced pressure. After repeating the process several times the solution is nucleated with seed crystals of the 1,4-lactone. - 10 -
Morrison and Perry (1966) oxidized a number of aldoses to aldonic acids and then converted them to their 1,4-lactones by the following procedure. The solutions were concentrated to dryness by distillation under reduced pressure below 40°C. The residues were dissolved in 2N
HCl and the solutions were distilled under reduced pressure. At the end of the distillation the flasks were immersed for two minutes in a boiling water bath whilst the last traces of volatile materials were removed under vacuum. Kjolberg and Veil an (1966) report the successful formation of 1,4-lactones without using any mineral acid. They prepared D-gluco- heptono-y-lactone from D-glucose by heating it in a boiling water-bath for several hours with sodium cyanide. Sodium ions were removed with a cation exchange resin and the solution was concentrated to a small volume under vacuum. The lactonization was achieved by heating the syrup to
100°C for 27 hours with mechanical stirring. Perry and Hulyalkar (1965) converted aldonic acids to the corresponding 1,4-lactones by treatment with concentrated HCl and evaporation to dryness. The final product was kept for 5 minutes at 100°C in vacuum. Petersson and others (1967a) successfully used Perry and Hulyalkar's method for the lactonization of a number of aldonic acids. Perry and others (1969) prepared heptono 1,4-lactones by concentrating the heptonic acid-lactone mixture in solution to near dryness, treating it with few drops of 2N HCl and then reconcentrating to dryness under reduced pressure, below 60°C. - n -
1.4 MASS SPECTROMETRY OF HEPTONOLACTONES
The TMS derivatives of some tetrano, pentono, hexono and heptono- lactones were subjected to mass spectrometric studies by Petersson and others (1967b). A weak molecular ion peak (M) was recorded for a-D-
Glucoheptonic acid y-lactone at m/e 568. The ion M-15 obtained when one methyl group is split off, was recorded at m/e 553. The upper part of the mass spectrum also indicated the occurrence of M-43 (m/e 525) and M-105 (m/e 463) fragments. The base peak corresponding to the
(CH^Si-ion occurred at m/e 73 for all lactones. Ions of m/e 217, 204,
189 and 147 were also recorded for all the lactones investigated.
1.5 PHASE TRANSITIONS
The recrystallization of calcium glucoheptonate may be due to a phase transition. Phase transitions from an unstable or metastable solid state to a stable state are usually of the following types:
(i) change from an amorphous to a crystalline form
(ii) change from an anhydrous to a hydrated or solvated form
(iii) change from a metastable polymorphic modification to a
stable form.
One of the first steps in formulating a solution is to determine the solubility of the drug in the vehicle (Haleblian and McCrone, 1969).
If the solubility is determined using a metastable form of the drug and the concentration of the drug in the formulation exceeds the equilibrium solubility of the stable form, a thermodynamically unstable preparation - 12 -
results. Solutions that are supersaturated with respect to the stable form of the drug may remain in this state for a long period of time.
Chance nucleation of the stable form, however, can result in crystallization until equilibrium is reached with the stable form. Certain phase transi• tions of pharmaceutical interest . which have been shown to result in recrystallization or changes in solubility and dissolution rate will be briefly reviewed.
1.5.1 Amorphous-crystalline transitions
Mullrhs and Macek (1960) identified two forms of novobiocin, one of which was crystalline and the other amorphous. When excess of solid
(< 10 u size) was shaken in 0.1 M HC1 at 25°C., the amorphous form was at least 10 times more soluble than the crystalline form. The difference in solubility was found to favor the absorption of the amorphous solid from the gastrointestinal tract. Unless special precautions are taken to maintain the solid in suspension in the amorphous state by the addition of materials to suppress crystallization, amorphous novobiocin slowly converts to a crystalline form. The formulation becomes less and less absorbable and finally loses its therapeutic effect.
Florence and Salole (1975) showed that comminution of crystalline digoxin resulted in the appearance of an amorphous phase. Using digoxin from different sources they observed increases in equilibrium solubili• ties between 7 and 118 percent due to the conversion from the crystalline to the amorphous phase. Black and Lovering (1978) attempted to determine the dissolution rate and apparent equilibrium solubility of digoxin samples differing in degree of crystall.tnf.ty,hoping to relate these properties, but found instead that rapid recrystallization took place. - 13 -
Chiou and Kyle (1979) attempted to determine the effect of trituration on the equilibrium solubility of digoxin but in some samples the equilibrium solubility was not affected. From dynamic solubility studies they concluded that the absence of solubility enhancement was due to conversion of the higher energy amorphous form to the more stable, lower energy crystalline form during the experimental period.
1.5.2 Hydrate-anhydrous form transitions
Haleblian and others (1972a) studied the interconversion of seven solid phases of fluprednisolone which included one tert-butylamine disolvate, two monohydrates ( a and 6 ), three anhydrous (Forms I, II and III) and one amorphous phase. All crystalline phases were converted to the a- monohydrate upon suspension in water. When the in vitro dissolution rates were compared (Haleblian and others, 1972b), the a-monohydrate was found to have the lowest rate of dissolution followed by the B-monohydrate.
Their studies suggest that the a-monohydrate is thermodynamically the most stable form. Ravin and others (1970) compared the solubility and dissolution rate of an anhydrous (Form I) and a monohydrate .(Form I*) form of an experimental antihypertensive. The anhydrous Form I dissolved much faster than the corresponding hydrated Form I*. Moreover, in aqueous suspension Form I readily forms the hydrate (Form I*).
Moustafa and others (1974) prepared two polymorphs, two hydrates, two solvates and an amorphous form of succinylsulfathiazole. On suspension in water, transformation to the dihydrate Form II occurred in all instances.
In a continued study, the same workers (Moustafa and others, 1975) examined the effect of various additives on the rate of transformation of the - 14 -
metastable anhydrous Form I to the water-stable Form II in aqueous suspen• sions. It was concluded that the formulation of physically stable suspen• sions of succinylsulfathiazole would best be achieved using water-stable
Form II or alternatively, including an efficient transformation retardant like methyl cellulose with Form I.
1.5.3 Polymorphic transitions
Aguiar and Zelmer (1969) compared the equilibrium solubilities of two polymorphic forms of chloramphenicol palmita'te (Forms A and B) and found that Form B was four times more soluble than Form A. The increased equilibrium solubility of this form was predominantly attributed to the- higher free energy content of this form. Clements and Popli (1973) compared the maximum concentration attained in solution (not equilibrium solubility) of two forms of meprobamate. The unstable form (Form II) was found to be twice as soluble as the stable form (Form I). However, after a certain period of time the concentration of Form II began to decrease, reaching an equilibrium value corresponding to the solubility of Form I.
Matsuda and others (1980) obtained four polymorphs of phenylbutazone by a spray drying method. The solubility of one of the metastable forms was found to be 1.5 times higher than that of the stable form. - 15 -
2. EXPERIMENTAL
2.1 APPARATUS
Autoclave, AMSCO general purpose, American sterilizer.
Atomic absorption spectrophotometer, model AA-5, Varian Techtron.
Cahn electrobalance, Gram, Ventron Coporation.
Constant temperature bath, Magni Whirl, Blue M Electric Company.
Differential scanning calorimeter with effluent gas analyzer, DSC-1B, Perkin-Elmer.
Freezer (-76°C), UC 105, Kelvinator.
Freeze-drying unit, Virtis company.
Gas chromatograph with a flame ionization detector, model 5830 A, Hewlett Packard and a GC terminal, model 18850 A, Hewlett Packard.
Gas chromatographic syringe, Hamilton.
Infrared spectrophotometer, Unicam SP 1000, Pye Unicam.
Mass spectrometer, MAT 111, coupled to a gas chromatograph, model 5700 A, Hewlett Packard and a computer, model 620/L, Varian. pH meter, model 26, Radiometer.
Sterifil filtration system, Millipore.
Vacuum pump, Vac Torr S 35, General Electric.
Vials (teflon-silicone screw-capped), Pierce.
X-ray diffractometer, wide angle, Philips. - 16 -
2.2. MATERIALS
Amberlite IR-120 ion-exchange resin, Mai 1inckrodt.
*Ca1cium glucoheptonate, Givaudan (supplied by May and Baker).
*Calcium glucoheptonate, Italsintex.
Calcium glucoheptonate USP (pure a epimer), Pfanstiehl.
Calcium a-B-glucoheptonate (calcium glucoheptonate, a-8 mixture), Pfanstiehl.
3% cyanopropylmethyl silicone (SILAR IOC) on Chromosorb W(HP) 100-120 mesh Applied Science.
3% cyanopropylphenylmethyl silicone (OV-225) on Chromosorb W(HP) 100-120 mesh, Western Chromatography.
Dimethylsulfoxide, BDH.
95% v/v Ethyl alcohol, commercial grade and redistilled.
a-D-Glucoheptonic acid y-lactone, Aldrich.
Hydrochloric acid, ACS grade, Allied Chemical.
Methanol, ACS grade, Caledon.
3% methyl silicone (OV-101) on Chromosorb W (HP), 100-120 mesh, Western Chromatography.
3% phenylmethyl silicone (OV-17) on Chromosorb W (HP), 100-120 mesh, Western Chromatography.
Methyl stearate, Matheson Coleman and Bell.
Sodium hydroxide, ACS grade, Fischer.
Sodium hydride/oil dispersion, Aldrich.
Sucrose, analytical reagent, BDH.
Trimethylsilylimidazole (TSIM), Pierce.
Trimethylsilylimidazole in pyridine (TRISIL Z), Pierce.
* gift from Stanley Drug Products. - 17
A mixture of trimethylsilylimidazole (TSIM), N,0-bis(trimethylsilyl) acetamide (BSA) and trimethylchlorosilane (TMCS)-TRISIL 1TBT\ Pierce.
Water, distilled. - 18 -
2.3 STABILITY STUDIES OF CALCIUM GLUCOHEPTONATE SOLUTIONS
The commercial formulation marketed by Stanley Drug Products contained
26.7% w/v calcium glucoheptonate in water plus a number of additives.
Since these additives may alter the precipitation behavior, preliminary studies involved the preparation of simple solutions containing 26.7% w/v calcium glucoheptonate in water.
Calcium glucoheptonate from the following manufacturers was used in the studies:
(i) Givaudan (through May and Baker)
(ii) Italsintex
(iii) Pfanstiehl. The two grades of material supplied by Pfanstiehl
were:
(a) USP grade (pure a form)
(b) a - 3 mixture
2.3.1 Solutions made from calcium glucoheptonate after heating at 120°C
Muller and others (1979) reported that solutions prepared after heating the calcium glucoheptonate powder at 115-120°C often resulted in stable solutions. Thus, calcium glucoheptonate was heated at 120°C for 12 hours, then cooled in a desiccator and used to prepare solutions. - 19 -
2.3.2 Heat treatment of calcium glucoheptonate solutions
(a) solutions were prepared and then heated at 80°C in a water
bath for 30 minutes.
(b) solutions were autoclaved at 121°Cfor 20 minutes.
2.3.3 Membrane filtration of calcium glucoheptonate solutions
Solutions were filtered through a 0.2 ym membrane filter (Millipore) in a sterilized Sterifil Filtration System. The results of these studies are presented in Table I.
2.4 CHARACTERIZATION OF CALCIUM GLUCOHEPTONATE AND THE PRECIPITATE
OBTAINED FROM SOLUTIONS OF CALCIUM GLUCOHEPTONATE
The calcium glucoheptonate was dried under vacuum at 60°Cfor 16 hours.
The precipitate obtained from solutions of calcium glucoheptonate was dried under the following conditions:
(1) at room temperature under vacuum to a constant weight. This
material was used for all the studies involving the charac•
terization of the precipitate.
(2) for 2 hours at 76°C under vacuum (5 mm). This was used in DSC
studies.
(3) for 46 hours at 80°C under vacuum (5 mm). This' material was subjected
to X-ray, IR, and solubility studies.
2.4.1 Elemental analysis
The elemental composition (carbon, hydrogen and oxygen) of calcium - 20 -
glucoheptonate USP (Pfanstiehl) and the precipitate obtained from the solution of calcium glucoheptonate USP (Pfanstiehl) were determined by
Canadian Microanalytical Service Ltd., Vancouver. The calcium content was determined in our laboratory by the USP assay method (USP XX, 1980) of calcium glucoheptonate.
2.4.2 Thermal analysis
A differential scanning calorimeter equipped for effluent gas analysis was used for performing thermal analysis. The materials were ground in a glass mortar and pestle and 1-5 mg samples were weighed with a Cahn electro- balance directly into aluminium volatile sample pans. Scans were made at 10°/minute using closed pans and pans with a 0.1-0.2 mm pinhole.
Vaporization of the water of hydration from the pans with a pinhole was detected using the effluent gas analyzer and was estimated quantitatively by weighing the pan after the appearence of the endothermic peak.
2.4.3 Infrared (IR) spectra
A. Preparation of solid samples
Pellets were prepared after mixing 5 mg of material with 200 mg of potassium bromide.' in a glass mortar and pestle.
B. Preparation of solutions
One mg of the sample was dissolved in 40 mL chloroform and this solution was used for obtaining the IR spectra. - 21 -
2.4.4 Heat of solution
About 500 mg of the sample was accurately weighed into the sample cell of a solution calorimeter. This was dissolved under controlled conditions in 100 g of water contained in a double walled glass vessel. Throughout the reaction, the temperatures were sensed by a thermistor and recorded on a strip chart recorder.
2.4.5 X-ray diffraction
This was carried out with Ni filtered CuKa X-rays, 40 kV, 15 ma, over a range of 20 from 10° to 60° at 1° 29/min (2 second count).
Approximately 300 mg of the ground sample was used.
2.4.6 Equilibrium solubility
An excess of the sample was added to 50 mL of water and the mixture was kept in a water bath with a shaking arrangement. The water-bath was maintained at 30°C. Samples were taken periodically, filtered and analyzed by the USP assay method of calcium glucoheptonate. The process was continued until equilibrium had been attained.
2.5 DEVELOPMENT OF A GAS CHROMATOGRAPHIC (GC) METHOD FOR ESTIMATING
THE PROPORTIONS OF THE a AND g EPIMERS IN CALCIUM GLUCOHEPTONATE
2.5.1 Selection of a substance for preliminary studies
Since carbohydrates are non-volatile compounds, they cannot be analyzed by GC unless voltaile derivatives are first formed (Laker, 1980).
Hence the first objective was the preparation of a stable, volatile - 22 -
derivative of calcium glucoheptonate.
The formation of an incomplete derivative is a problem often encoun• tered and this is readily identified by the appearance of multiple peaks
in the chromatogram. For the preliminary studies it was necessary to use a known pure single compound which would give a single peak if completely derivatized. Calcium glucoheptonate could not be used because it was suspected that it might be a mixture of the a and 3 forms,The y-lactone of a-D-glucoheptonic acid is commercially available and was used for the preliminary derivatization reactions.
2.5.2 Preparation of methyl derivative
The method used was a modification of that reported by Leclereq and Desiderio (1971).
A. Preparation of sodium methylsulfinylmethide
An amount of sodium hydrtde/oil dispersion containing 25 mg sodium hydride was rinsed 3 times with anhydrous ether. 1 mL of dry dimethyl-
sulfoxide (DMSO) was added and the suspension heated under nitrogen, until evolution of hydrogen ceased.The resulting clear solution was stored under nitrogen in a refrigerator.
B. Derivative formation
To 200 yg of the y-.lactone of a-D-glucoheptonic acid, 100 uL of DMSO was added. A stream of nitrogen was passed through the tube. Then 20 uL of sodium methylsulfinylmethide was added, the vial was set aside for
15 minutes and 40 uL of methyl iodide was added. The tube was immersed - 23 -
in an oil bath and heated at 40°Cfor 30 minutes. The space above the liquid
in the tube was flushed with nitrogen, closed with the screw cap, sealed with teflon tape and kept in an oven at 58°Cfor 1 hour. The reaction was
terminated by the addition of 1 mL of water to the contents of the tube.
The methylated product was extracted by shaking with 1 mL of chloroform and removing the water layer. The chloroform layer was washed 2 times with
1 mL of water and the chloroform was evaporated off in a stream of nitrogen.
The residue was redissolved in 100 uL of chloroform.
When 5 uL of the above solution was injected into a gas chromatograph with an OV-225 column it resulted in multiple peaks indicating that methyla-
tion was incomplete.
2.5.3 Preparation of trimethylsilyl derivative
A number of silylating agents commercially available from Pierce
Chemical Company, U.S.A. were investigated as potential derivatizing agents prior to gas chromatography on a OV-225 column.
A. N-Trimethylsilylimidazole (TSIM)
Ffve mg of sample was dissolved in 0.1 mL pyridine in a 1.0 mL vial.
Then 0.4 mL of TSIM was added and the vial vortexed. The vial was heated at 60°Cfor 30 minutes. Five uL of the above sample was injected into the gas chromatograph. This resulted in multiple peaks indicating incomplete trimethylsilylation.
B. Mixture of trimethylsilylimidazole, N,0-bis(trimethylsi1yl) acetamide,
and trimethylchlorosilane (TRISIL 'TBT')
'Five mg of sample was derivatized using TRISIL 'TBT' as above and - 24 -
5 uL of the resulting solution was injected into the gas chromatograph.
This also resulted in multiple peaks thereby indicating incomplete derivatization.
C. Trimethylsilylimidazole in pyridine (TRISIL Z)
Since this reagent is formulated in pyridine, 5.0 mg of sample was directly dissolved in 0.5 mL of TRISIL Z. This solution was warmed at
60°C for 30 minutes and then 5 uL was injected into the gas chromatograph.
This resulted in a single peak, thereby indicating apparently complete trimethylsilyation. The solution was injected into the gas chromatograph containing columns of varying polarities namely:
(i) 3% 0V-101 on Chromosorb W(HP) 100-120 mesh
(ii) 3% OV-17 on Chromosorb W(HP) 100-120 mesh
(iii) 3% OV-225 on Chromosorb W(HP) 100-120 mesh
(iv) 3% SILAR 10 C on Chromosorb W(HP) 100-120 mesh
In each case there was only one peak thereby confirming that the trimethyl- silylation was complete.
2.5.4 Preparation of the trimethylsilyl derivative of calcium glucoheptonate
To 5 mg of calcium glucoheptonate (Pfanstiehl, a-3) 0.5 mL of TRISIL
Z was added and vortexed and 5 uL was injected into the gas chromatograph with an OV-225 column. This did not result in any peaks. The chromato• graphy was unsuccessful presumably due to the non-volatile nature of the calcium salt. Hence it was decided to pass a solution of calcium gluco• heptonate through a cation exchange resin and exchange the calcium for hydrogen to form glucoheptonic acid. About 10 g of the cation exchange resin
Amberlite IR-120 was first washed with IM NaOH solution. It was then - 25 -
repeatedly washed with water, then with 1 Nl formic acid, again with water
and loaded into a glass column. One gram of calcium glucoheptonate
(Pfanstiehl, a-B) was dissolved in deionized distilled water and passed (<5 ppm)
through the ion exchange column. This solution was analyzed for calcium
in the atomic absorption spectrophotometer. Negligible levels of calcium
were observed thereby confirming the efficiency of the ion exchange
process. This solution was frozen by storing at -76°Cin a freezer. The
solution was freeze-dried to a constant weight and about 5 mg of the
freeze dried material was transferred to a 1 mL vial and 0.5 mL of
TRISIL Z was added. The vial was vortexed and heated at 60°Cfor 2
hours. Then 5 uL was injected into the gas chromatograph with an OV-225
column. This gave four peaks presumably due to a-p-glucoheptonic acid, B-
D-glucoheptonic acid, and the corresponding lactones which result from
lactonization of the acids in aqueous solution (Isbell and Frush, 1963).
The formation of both 1,4-lactones as well as 1,5-lactones are possible.
These reactions are presented in schemes IA and IB.
In order to positively identify these four peaks it would be necessary
to have the following pure reference compounds:
(i) a-D-glucoheptonic acid
(ii) B-D-glucoheptonic acid
(iii) y-lactone of a-D-glucoheptonic acid
(iv) y-lactone of B-D-glucoheptonic acid
(v) S-lactone of a-D-glucoheptonic acid
(vi) S-lactone of B-D-glucoheptonic acid
Unfortunately,only the y-lactone of a-D-glucoheptonic acid is commercially available. - 26 -
c = 0 COOH 0 = C 1 1 i - OH H - C - OH H - OH - c I - c 1 i 1 1 - OH H - OH H - OH - c - c - c -H90 -H20 1 1 1 1 - H OH 1 - H OH 1 - H — c - c - c +H20 +H20 1 1 1 1 - OH H 1 - OH H 1 - c - c - c 1 1 1 - OH H 1 - OH H 1 - OH - c - c - c
CH2OH CH20H CH20H
1,4-lactone of a-D-glucoheptonic 1,5 1actone of a-D-glucoheptonic acid a-D-glucoheptonic acid acid (f-lactone of (6-lactone of a-D-glucoheptonic a-D-glucoheptonic acid) acid)
Scheme IA. Lactonization of a-D-glucoheptonic acid.
0 COOH 0 I OH C - H OH - C - H OH C - H. •H20 I 1 •H20 I H C - OH H - C - OH H C - OH I I +H20 I +H20 I c - H OH - C - H OH C - H I I I c - OH H - C - OH H C I | I OH H - C - OH H C - OH c - I 1 CH20H CH2OH CH2OH
1,4-lactone of 3-D-glucoheptonic 1,5-lactone of 6-D-glucoheptonic acid B-D-glucoheptonic acid acid
Scheme IB. Lactonization of g-D-glucoheptonic acid. - 27 -
2.5.5 Lactone formation
In the presence of hydrochloric acid aldonic acids are converted into their corresponding y-lactones (1,4-lactones) (Perry and Hulyalkar, 1965).
Hence the number of peaks occurring in the gas chromatographic analysis can be reduced by treating the eluate from the ion exchange column with hydrochloric acid in order to convert the glucoheptonic acid/lactone mixture completely to the lactones. Treatment of the acid-lactone mixture with hydrochloric acid also avoids the possible complication due to the formation of 1,5-lactones (Isbell and Frush, 1963; Perry and Hulyalkar,
1965).
A stock solution of the freeze-dried eluate was prepared in methanol and an accurately measured volume (containing about 0.25 mg of gluco- heptonic acid/lactone mixture) was transferred to a 1 mL vial. The methanol was evaporated off under reduced pressure, 50 uL of concentrated
HCl was added and the solution vortexed. The HCl was evaporated off under reduced pressure. A further 50 uL of concentrated HCl was added and this process repeated three more times. Finally, 100 yL of TRISIL Z was added, the solution heated at 60°C for 30 minutes, vortexed and injected into the gas chromatograph with an OV-225 column. This resulted in two peaks (Fig. 5).
A control experiment using the TMS derivative of the Y-lactone of a-D-glucoheptonic acid reference material (Fig. 6) gave a single peak having the same retention time as the peak 2 (Fig. 5) of the sample, thereby suggesting that this second peak is due to the TMS derivative of the y-lactone of a-D-glucoheptonic acid. However, evidence was necessary to establish that the first peak was due to the TMS derivative of the Y-Tactone of 3-D-glucoheptonic acid. - 28 -
1. B-D-glucoheptonic acid Y-lactone
2. a-D-glucoheptonic acid Y-lactone
r T 1 1 0 5 10 15
MINUTES
Fig. 5. Chromatogram of the TMS derivatives of the Y-lactones of a and B-D-glucoheptonic acids.
Chromatographic conditions: column, 3% OV-225 on Chromosorb W (1.8 m x 4 mm); injection temperature, 250°C; detector temperature, 250°C; column temperature, 200°C; carrier gas (helium) flow rate 30 mL/min. Fig. 6. Chromatogram of the TMS derivative of the y-lactone of a-D-glucoheptonic acid (reference material).
Chromatographic conditions: column, 3% OV-225 on Chromosorb W (1.8 m x 4 mm); injection temperature, 250°C; detector temperature, 250°C; column temperature, 200°C; carrier gas (helium) flow rate 30 mL/min. - 30 -
2.5.6 Gas chromatography^mass spectrometry (GC-MS)
The procedure for preparation of the sample was the same as elaborated
in section 2.5.5 (lactone formation). A 1.25 m, 2.5 mm i.d. glass column
packed with 3% OV-225 on Chromosorb W was used for the GC-MS studies.
The other experimental conditions were:
injection temp. 250°C
column temp. 200°C
separator temp. 250°C
carrier gas flow rate: 30 mL/min
Beam energy 70 eV
When the sample was injected, it resulted in two peaks (as in Fig. 5). The
two GC peaks had similar mass spectral patterns. The fragmentation pattern
summarized in Scheme 2 had the following characteristics:
(i) a molecular ion peak (M+) recorded at m/e 568 in agreement
with that calculated for the fully trimethylsilylated
derivative.
(ii) the ion M-15 (obtained when one methyl group is split off) at
m/e 553.
(iii) the ion M-43 at m/e 525.
(iv) the ion M-105 observed at m/e 463 due to the loss of CH3 and
TMSOH groups.
The reference material i.e. the y-lactone of a-D-glucoheptonic acid, when subjected to GC-MS analysis had the same retention time and frag• mentation pattern as the peak 2 in Fig. 5. Hence the identity of this peak, as the TMS derivative of the y-lactone of a-D-glucoheptonic acid - 31 -
Peak 1 Peak 2
0 C = 0 I TMS 0 - C H H - C - 0 TMS I H - C - 0 TMS H - C 0 TMS I I C - H C H I a 0 TMS H - C 0 TMS I H - C - 0 TMS H - C - 0 TMS I
2 CH 0 TMS CH20 TMS
TMS derivative of y-lactone TMS derivative of y-lactone of of 3-D-glucoheptonic acid a-D-glucoheptonic acid
M (MW 568) M (MW 568)
-CH, -CH2CH2CH2 -CH, CH2CH2CH2
m/e 553 m/e 525 m/e 553 m/e 525
TMS OH TMS OH
m/e 463 m/e 463
Scheme 2. Fragmentation scheme of the TMS derivatives of
the y-lactones of a-D-qlucoheptonic acid
and g-D-glucoheptonic acid. - 32 -
was confirmed.
Since GC peaks 1 and 2 (Fig. 5) have different retention times but mass spectral patterns with identical m/e values at the upper region of the spectrum, the chemical structures of the two compounds must be very similar. Since the identity of peak 2 was established, peak 1 was ascribed to the TMS derivative of the y-lactone of B-D-glucoheptonic acid.
2.5.7 Selection of internal standard
Glucose, mannose and sucrose were chosen as compounds for investiga• tion as possible internal standards. About 5 mg of each of these substances was taken in a 1 mL vial and 0.5 mL of TRISIL Z added. The vial was vortexed and heated at 60°C for 30 minutes. Then 5 uL was injected into the gas chromatograph with an OV-225 column. The TMS derivatives of glucose and mannose had very short retention times coming off almost with the solvent. On the other hand, the TMS derivative of sucrose had a longer retention time than the compounds under investigation i.e. the
TMS derivatives of the y-lactones of a and B-D-glucoheptonic acid.
Hence sucrose was used as the internal standard. It is available in a highly purified form and the preparation of stock solution posed no problem.
2.5.8 Optimization of GC conditions
The preliminary experiments were carried out on a 1.8 m glass column,
4 mm i.d. packed with 3% OV-225 on Chromosorb W under the following conditions: - 33 -
Injection temp. 250°C
Column temp. 200°C
Detector (F.I.D.) temp. 250°C
Carrier gas (helium) flow rate: 30 mL/min
A. Selection of stationary phase
The following stationary phases were investigated for possible use:
(i) 3% OV-17 on Chromosorb W'(HP) 100-120 mesh
(ii) 3% OV-101 on Chromosorb W(HP) 100-120 mesh
(iii) 3% OV-225 on Chromosorb W(HP} 100-120 mesh
(iv) 3% SILAR 10 C on Chromosorb W 100-120 mesh
Of the four phases tested, the most satisfactory separation was achieved with the OV-225 phase. A 1.8 m, 4 mm i.d. glass column packed with 3%
OV-225 on Chromosorb W was used for subsequent GC work.
B. Temperature programming
When the TMS derivative of sucrose was chromatographed at an iso• thermal column temperature of 200°, it had a retention time of about
17 minutes. Because of the relatively long retention time, the peak due to sucrose was quite broad. The column temperature could not be increased any further because the retention times of the TMS derivatives of y-lactones of a and B-D-glucoheptonic acids were close to each other
(Fig. 5). Hence temperature programming was done. The oven temperature was kept at 200° for the first 10 minutes, then it was increased to 215° at 5°/minute. The retention time of sucrose was reduced to about 14.8 - 34 - minutes and the peak shape was also greatly improved (Fig. 7).
C. Injection temperature
Conventionally the injection temperature is 50° to 100°C higher than
the oven temperature (Burchfield and Storrs, 1962). The injection tempera•
ture was. varied from 230° to 270°C and no difference in the response of a-D-glucoheptonic acid y-lactone and B-D-glucoheptonic acid y-lactone was observed. Variation in injection temperature did not produce any difference
in the proportions of the a and $ epimers. Hence the injection temperature was set at 250°C.
D. Detector temperature
The detector temperature was also set at 250°. A variation in the detector temperature of ±20° did not produce any difference in the response of y-lactones of a-D-glucoheptonic acid and B-D-glucoheptonic acid. There was no difference in the proportions of these two compounds due to a change
in detector temperature.
E. Optimization of reaction time
It was necessary to determine the time required for the apparently complete derivatization of y-lactones and sucrose. The y-lactones and
sucrose undergo the same derivatization reaction. Hence it was first necessary to determine the time taken for the optimal trimethylsilyation of
sucrose. For this purpose methyl stearate was chosen as the internal standard
because it does not undergo the trimethylsilylation reaction. A mixture of sucrose and methylstearate were dissolved in TRISIL Z, and the area ratio of sucrose to methylstearate was determined. This mixture was then 35
1. B-D-glucoheptonic acid y-lactone
2. a-D-glucoheptonic acid Y-lactone
3. sucrose
10 15 ~20
MINUTES
Fig. 7. Chromatogram of the TMS derivatives of the y lactones of a and g-D-glucohetponic acids with the TMS derivative of sucrose as the internal standard.
Chromatographic conditions: column, 3% OV-225 on Chromosorb W (1.8 m x 4 mm); injection temperature, 250°C; detector temperature, 250°C; column temperature, 200°C (10 min.) to 215°C at 5°C/min; carrier gas flow rate 30 mL/min. - 36 - heated at 60°C for 15 and 30 minutes and the area ratios were again determined.
There was no difference in the area ratio indicating that the derivatization reaction of sucrose was instantaneous at room temperature. Now using sucrose as the internal standard, a-D-glucoheptonic acid y-lactone and
B-D-glucoheptonic acid y-lactone were subjected to a similar series of experiments. Again it was observed that the apparently complete trimethylsi- lation of the two lactones -was accompli shea* instantaneously without heating.
2.6 PREPARATION OF STANDARD CURVE OF THE y-LACTONE OF g-D-GLUCOHEPTONIC
ACID
About 60 mg of the y-lactone of a-D-glucoheptonic acid was accurately weighed and dissolved in sufficient methanol to make up the volume to
100 mL. The following amounts of the lactone (as methanolic solution) was transferred to 1 mL vials - 30, 60, 90, 120, 150 and 180 yg. Sixty
-yg of sucrose (as a solution in pyridine) was added to each of the vials.
Then 200 yL of TRISIL Z was added to each vial, the vials vortexed and
5 yL was injected into the GC. The standard curve was plotted (Fig. 7a).
Each point was the mean value obtained with 5 or more injections.
2.7 DETERMINATION OF THE PROPORTIONS OF a AND g EPIMERS IN COMMERCIAL
SAMPLES OF CALCIUM GLUCOHEPTONATE
The proportions of the a and B epimers was determined in the following commercial samples:
(i) calcium glucoheptonate (Givaudan)
(ii) calcium glucoheptonate (Italsintex) - 37 -
WEIGHT RATIO
Fig. 7a.. Standard curve of the y-lactone of a-D-glucoheptonic acid. - 38 -
(iii) calcium gluceptate USP (Pfanstiehl)
(iv) calcium a-B-glucoheptonate(Pfanstiehl)
About 1 g of calcium glucoheptonate was dissolved in water and passed through cation (Amberlite IR-120 H) exchange resin. The eluate was freeze- dried to constant weight and about 70 mg was accurately weighed and dissolved in sufficient methanol to 100 mL. Then 150, 200 and 250 uL of this methanolic solution were transferred to 1 mL vials and the methanol was evaporated off under reduced pressure. Next 100 uL of concentrated
HCl was added to each vial, the vials were vortexed and the hydrochloric acid was evaporated off under reduced pressure. The addition and evaporation of HCl was repeated three more times. About 60 mg of sucrose was dissolved in pyridine and the volume made up to 100 mL.
From this solution, 100 uL was added to each of the vials (internal standard) followed by 200 uL of TRISIL Z. The vials were vortexed and a five yL sample was injected into the GC.
2.8 DETERMINATION OF THE PROPORTION OF a AND p EPIMERS IN THE PRECIPITATE
OBTAINED FROM COMMERCIAL SAMPLES OF CALCIUM GLUCOHEPTONATE
The procedure followed was the same as discussed in section 2.7.
The precipitate was subjected to GC-MS studies and the procedure followed was the same as in section 2.5.6. The fragmentation pattern was the same as in Scheme 2. - 39 -
3. RESULTS AND DISCUSSION
3.1 STABILITY STUDIES OF CALCIUM GLUCOHEPTONATE SOLUTIONS
The results presented in Table I indicate that the Pfanstiehl (USP) material has the lowest stability in solution while the Pfanstiehl (a - B) sample has the greatest stability in solution. Filtration increases the time taken for precipitation to occur while autoclaving results in stable solutions which have not precipitated in more than a year.
3.2 CHARACTERIZATION OF CALCIUM GLUCOHEPTONATE AND THE PRECIPITATE
OBTAINED FROM SOLUTIONS OF CALCIUM GLUCOHEPTONATE
3.2.1 Elemental analysis
The elemental composition of calcium glucoheptonate USP (Pfanstiehl) and the precipitate obtained from a solution of calcium glucoheptonate given in Table II indicate that both the initial material and the precipitate have nearly identical elemental compositions.
3.2.2 Thermal analysis
The calcium glucoheptonate available from each source was subjected to differential scanning calorimetric (DSC) studies as received. On heating to 180°C there were neither exothermic nor endothermic peaks showing that the original materials were anhydrous and that no polymorphic transitions occurred in the experimental temperature range. However, the precipitate showed a single endothermic peak around 110°C with a weight loss following - 40 -
Table I. Stability of calcium glucoheptonate
(26.7 percent w/v) in aqueous solution.
Time for precipitation to occur (days) Commercial Source
Treatment Givaudan Italsintex Pfanstiehl USP a - 3
Control9 2 8 1-
Solid heated 120°C x 12 hours 3 3 1 ND
Solution heated 85°C x 30 min. 13 7 1 ND
Solution autoclaved l"? TOT v On min
Filtered0 210 240 2
Filtered0 and autoclaved - - -
- = no precipitation
ND = not done a = solution prepared by dissolving solid in water at room temperature; frequently contaminated with microbial growth b = causes caramelization c = 0.22 ym membrane filter (Millipore)
Studies carried out in June, 1980. - 41 -
Table II. Elemental composition of calcium glucoheptonate
and the precipitate obtained from the solution
of calcium glucoheptonate.
calcium glucoheptonate USP Precipitate3 (Pfanstiehl)
Theoretical Experimental
Carbon 34.3 % 33.4 32.6 %
Hydrogen 5.34 % 5.48 % 5.67 %
Calcium 8.17 % 8.12 % 7.88 %
Oxygen 52.2 % 40.lb % 42.3b %
Dried under vacuum at room temperature to constant weight.
The low values (compared with theoretical) are not explainable. - 42 -
the endothermic peak suggesting that the precipitate might be a hydrate.
When the precipitate was dried under vacuum at 76°Cfor 2 hours and the
DSC scan repeated, the endothermic peak disappeared. Attempts were made to determine the number of molecules of water of crystallization by quantitating the weight loss after the appearance of the endothermic peak but the weight loss was highly variable and not consistent with any specific value for waters of crystallization. It is likely that the precipitate from aqueous solution consists of a mixture of stoi• chiometric hydrates and/or is a hydrate of variable composition.
3.2.3 IR spectra
A. Solid samples •
The IR spectra in KBr discs were determined for:
(i) calcium glucoheptonate (Givaudan)
(ii) calcium glucoheptonate (Italsintex)
(iii) calcium glucoheptonate (Pfanstiehl, a-8 )
(iv) calcium glucoheptonate USP (Pfanstiehl)
(v) precipitate obtained from the solution of calcium glucoheptonate
USP (Pfanstiehl) dried under vacuum at room temperature to
constant weight
(vi) precipitate obtained from the solution of calcium glucoheptonate
USP (Pfanstiehl) dried under vacuum at 80°Cfor 46 hours
Calcium glucoheptonate from all the suppliers showed a few large and poorly defined absorption bands (Fig. 8A). The precipitate which had been dried under vacuum at room temperature had a much more clearly - 43 -
Fig. 8. Infrared spectra of calcium glucoheptonate
and the precipitate (KBr disc).
A. calcium glcoheptonate USP (Pfanstiehl)
B. precipitate obtained from the solution of calcium glucoheptonate USP (Pfanstiehl) dried under vacuum at room temperature to constant weight
C. precipitate obtained from the solution of calcium glucoheptonate USP (Pfanstiehl) dried under vacuum at 80°C for 46 hours. - 44 -
3800 3500 2500 2000 Wawnumbai
Fig. 8A.
WWr*twifth nm
3800 3500 3000 2500 20O0 Wamnumber Fig. 8B.
Wav*numbai
Fig. 8C. - 45 -
Fig. 9. Infrared spectra of calcium glucoheptonate and
the precipitate (solution in chloroform).
A. calcium glucoheptonate USP (Pfanstiehl)
B. precipitate obtained from the solution of calcium glucoheptonate USP (Pfanstiehl) dried under vacuum at room temperature to constant weight
C. precipitate obtained from the solution of calcium glucoheptonate USP (Pfanstiehl) dried under vacuum at 80°C for 46 hours - 46 -
Wavelength fjm
Wavenumber Fig. 9A.
Wavelength fjm
Wavenumber Fig. 9B.
Wavenumber Fig. 9C. - 47 -
defined IR spectral pattern (Fig. 8B.). The broad absorption found near
3300 cm 1 is due to bonded OH stretching and the numerous bands in the region 1125-1000 cm-1 are due to the stretching of the C-0 bond. After drying under vacuum at 80°C for 45 hours the spectrum (Fig. 8C) lost its sharpness and appeared like the spectrum of the original calcium glucoheptonate i.e. Fig. 8A.
B. Solutions
Each of the samples investigated in section 3.2.3 A was dissolved in chloroform. The IR spectra of the solutions obtained were identical and superimposable. The spectra of the Pfanstiehl USP sample and the precipitate (dried under different conditions) were also superimposable as shown in Fig. 9.
3.2.4 Heat of solution
Heats of solution for calcium glucoheptonate obtained from different sources are presented in Table III.
3.2.5 X-ray diffraction studies
The following samples were subjected to X-ray diffraction studies.
(i) calcium glucoheptonate (Givaudan)
(ii) calcium glucoheptonate (Italsintex)
(iii) calcium glucoheptonate (Pfanstiehl, a-B )
(iv) calcium glucoheptonate USP (Pfanstiehl)
(v) precipitate obtained from a solution of calcium glucoheptonate
USP (Pfanstiehl) dried under vacuum at room temperature to
constant weight - 48 -
Table III. Heats of solution of calcium glucohepto•
nate samples.
Source Heat of solution kJ.mol ^
Italsintex 8.28 (1.98)a
Givaudan 10.0 (2.40)
Pfanstiehl (a,B) 16.2 (3.88)
Pfanstiehl (USP) 22.2 (5.31)
values in parenthesis are k.cal.mol
The heat of solution of the precipitate obtained from solutions of calcium glucoheptonate could not be determined because of its low aqueous solubility. - 49 -
Table IV. X-ray studies of the precipitate obtained from the.solution
of calcium glucoheptonate USP (Pfanstiehl).
Precipitate dried under Precipitate dried under vacuum vacuum at RT at 80°cfor 46 hours
a d(A) (I/I0) d(A) (I/I0) Relative Intensity Relative Intensity
7.82 7
7.49 21 7.56 7
- - 6.67 •8
- - 6.55 10
6.16 24 6.18 2
- - 5.85 10
- - 5.49 8
5.34 51 5.36 32
4.84 15 4.91 30
4.73 48 4.74 9
4.58 7 4.59 40
4.49 23 4.50 11
4.35 56 - -
4.33 >.*-ioo 4.34 > 100
- - 4.22 9
4.13 > 100 4.19 21
cont'd a d = distance between successive identical planes of atoms in a crystal - 50 -
Table IV/cont'd
Precipitate dried under Precipitate dried under vacuum vacuum at RT at 80°Cfor 46 hours
d(A) (I/I0) d(A) (I/I0) Relative Intensity Relative Intensity
- 4.03 6
- - 4.01 5
3.88 72 3.88 18
3.83 > 100 3.83 32
3.63 10 3.68 4
3.53 24 3.53 3
3.40 24 - -
3.33 > 100 3.33 22
3.26 10
3.22 22 3.23 8
3.14 16 3.14 7
3.07 47 3.07 7
3.01 15 3.03 7
2.94 58 2.94 9
2.89 10 - - 2.85 4 - -
2.80 18 - -
2.77 30 - -
2.69 7 2.68 8
cont'd - 51 -
Table IV/cont'd
Precipitate dried under Precipitate dried under vacuum vacuum at RT at 80°Cfor 46 hours
(I/I0) d(A) d(A) (I/I0) Relative Intensity Relative Intensity
2.67 55 - -
2.65 5 - -
2.59 10 - -
2.56 3 - -
2.51 3 - -
2.46 24 2.46 5
2.44 3 - -
2.39 68 2.39 20
2.34 31 2.34 10
- - 2.29 17
2.28 41 2.28 3
2.21 16 2.21 9
2.16 6 2.16 5
2.14 6 2.14 5
2.13 13 - -
2.10 5 2.10 5
2.09 3 2.09 5
- - 2.06 4
2.01 22 2.02 5
1.98 44 1.98 26
cont1 d - 52 -
Table IV/cont'd
Precipitate dried under Precipitate dried under vacuum vacuum at RT at 80°Cfor 46 hours
d(A) (I/I0) d(A) (^o) Relative Intensity Relative Intensity
1.92 17 1.92 5
1.90 18 1.90 4
1.88 3 -
1.85 8 -
1.83 5 -
1.81 9 1 .80 3
1.78 6 1.78 3
1.77 5 -
1.70 4 -
1.67 5 -
1 .62 3 -
1.59 2 -
1.55 5 -
1.45 4 -
1.40 4 -
1 .36 13 - 53 -
(vi) precipitate obtained from a solution of calcium glucoheptonate
USP (Pfanstiehl) dried under vacuum at 80°C for 46 hrs
The calcium glucoheptonate was found to be amorphous, irrespective of the source of the sample which disagrees with information received from Pfanstiehl that production of the amorphous form has been impossible in recent years (Holstein, 1980). On the other hand, the precipitate had a characteristic X-ray diffraction pattern thereby indicating its crystalline nature. When the precipitate was dried under vacuum at 80°C for several hours, there was a change in the X-ray diffraction pattern. Peaks at certain d values disappeared and new peaks appeared but there was a reduction in the total number of peaks (Table IV).
3.2.6 Equilibrium solubility
A. Calcium glucoheptonate
Attempts to determine the equilibrium solubility of calcium gluco• heptonate were unsuccessul. The material seems to be "infinitely" soluble in water. The viscosity of the solution increases dramatically as more and more solid goes into solution and shaking becomes increasingly less effective. Moreover in highly concentrated solutions recrystalliza- tion (except for Pfanstiehl ct-B mixture) occurs much sooner and there is no time for attainment of equilibrium.
B. Precipitate obtained from a solution of calcium glucoheptonate USP
(Pfanstiehl) dried under vacuum at room temperature to constant weight
This material was found to have a solubility of 2.5% w/v in water. - 54 -
C. Precipitate obtained from a solution of calcium glucoheptonate USP
(Pfanstiehl) dried under vacuum at 80°C for 46 hours
This material was found to have a solubility of 10% w/v in water.
3.3 IDENTIFICATION OF THE PRECIPITATE
Elemental analysis (Table II) showed that calcium glucoheptonate and the precipitate (dried under vacuum at RT to constant weight) have almost identical elemental compositions. The IR studies of solutions in chloroform revealed that the spectra of calcium glucoheptonate and the dried precipitate were superimposable (Fig. 9). Finally, the mass spectral pattern of calcium glucoheptonate (Scheme 2, p. 31) and the dried precipi• tate (p. 38) were identical. Hence it was concluded that the dried precipitate and the initial material were chemically identical. However, from the DSC studies (Section 3.2.2) it was concluded that the precipitate was liekly to be a hydrated form. The number of molecules of water of crystallization was variable so the precipitate is likely to be a mixture of hydrates or a hydrate of variable composition. Drying the precipitate under vacuum at 76°C for 2 hours resulted in the loss of water. Drying the precipitate under vacuum at 80°C for 46 hours not only resulted in the loss of water but also produced changes in the X-ray diffraction pattern (Table IV). It seems that when the hydrated form of calcium glucoheptonate loses its water, the crystal loses its lattice structure but again recrystallizes. This phenomenon could explain the change in the diffraction pattern which occurs on drying. - 55 -
3.4 POSSIBLE REASONS FOR PRECIPITATION
The precipitation of calcium glucoheptonate from solutions could be due to one or more of the following reasons:
(i) a change from an unstable modification to a stable form
(ii) the presence of seed crystals in the environment or in
the material which induce crystallization
(iii) differing proportions of the a and 8 epimers in the calcium
glucoheptonate obtained from various sources
3.4.1 Change from an unstable form to a stable form
X-ray studies (Section 3.2.5) indicated that the calcium glucohepto• nate was amorphous while the precipitate was crystalline. On the other hand, DSC studies (Section 3.2.2) revealed that calcium glucoheptonate is anhydrous but the precipitate is hydrated. Hence the change that is occuring is from an amorphous anhydrous material to a hydrated crystalline precipitate.
The precipitate has been identified as calcium glucoheptonate
(Section 3.3). Solubility studies (Section 3.2.6 A and B) indicate that calcium glucoheptonate is "infinitely" soluble while the precipitate
(dried under vacuum at room temperature to constant weight) is only 2.5% w/v soluble in water. This dramatic change in solubility could be due to one or other or both of these reasons:
(i) amorphous to crystalline transition
(ii) anhydrous to hydrate transition - 56 -
When the precipitate was rendered anhydrous by heating it at 80 °C for 46 hours under vacuum, there was a change in the X-ray diffraction pattern (Table IV). This suggested a change in the crystal lattice structure. Hence based on the studies carried out so far, it is not possible to isolate which of the above two factors is more responsible for this drastic change in solubility.
3.4.2 Presence of seed crystals inducing crystallization
The stability studies of calcium glucoheptonate solutions (Table I) indicate that membrane filtration exerts a stabilizing action on the solution. It can be postulated that the majority of the seed crystals are excluded by filtration which results in increased stability of the solution. Autoclaving could destroy the seed crystals and thereby explain the prolonged stability of autoclaved solutions. These results indicate that although the possibility of seed crystals inducing crystallization cannot be ruled out, the presence of seed crystals cannot be the only cause of solution instability for the following reasons:
(i) solution prepared using the Pfanstiehl (a-8) mixture are
stable. If seed crystals were the only causative factor
for instability, then the solutions prepared using Pfanstiehl
(a-g) should also have precipitated,
(ii) the results in Table I indicate that the time taken for
precipitation to occur depends on the source of calcium
glucoheptonate. Moreover, there is a wide variation in
the heat of solution values of calcium glucoheptonate
(Table III) obtained from different sources. These facts
suggest that the various samples of calcium glucoheptonate - 57 -
behave differently due to differences in their chemical
composition and/or physical properties.
3.4.3 Differing proportions of the a and B epimers in the calcium
glucoheptonate obtained from various sources
For testing this hypothesis it was necessary to:
(i) develop methods to identify the a and B epimers in the
commercial samples of calcium glucoheptonate
(ii) develop methods for estimating the proportions of the
a and B epimers in commercial samples
(iii) correlate the proportions determined with the observed
stability results
The analytical method for estimating the proportions of the a and 8 epimers has been elaborated in Section 2.5. The results in Table V indicate that there is a marked variation in the proportion of the a and B epimers in the samples supplied by different manufacturers. A correlation between the proportions of the a and 8 epimers and the stabil in solution (from Table I) is given in Table VI. These results show that there is a relationship between the stability in solution and the propor• tion of the a epimer. The Pfanstiehl USP sample which consisted of the a epimer (i.e. 100% a) precipitated from solution within one day. On the other hand, the Pfanstiehl (a - B) sample which contains 51.8% a form has been stable for more than a year. Thus the 8 form seems to stabilize the a form in solution and it is possible that there is a critical concen tration of the B f°rm necessary for stabilization.
The proportions of the a and 8 epimers in the precipitate obtained - 58 -
Table V. Determination of the proportion-' of the OK epimer
in various commercial samples of calcium gluco•
heptonate.
Source Proportion of the a epimer Mean Standard (percent) Deviation
concn.a concn. concn.
May & Baker 72.52 72.20 72.04 72.25 ±0.2444
Italsintex 71.88 71.78 71.84 71.83 ±0.0503
Pfanstiehl (a-B) 51.31 50.30 51.27 50.96 ±0.5719
Pfanstiehl USP 100.0 100.0 100.0 100.0 ±0.0000
aThe proportions in each concentration is the mean from 3 injections. - 59 -
Table VI. Relationship between the proportion of the
a epimer and the stability of calcium gluco•
heptonate in solution.
Source Proportion of g epimer Time for precipitation (percent) to occur (days)
Pfanstiehl USP 100 < 1
Italsintex 72.4 2
May & Baker 71.8 8
Pfanstiehl (a-3) 51.8 stable - 60 - from solutions of calcium glucoheptonate were determined (Table VII) and
the results indicate that there is an increase in the proportion of the a epimer in the precipitate when compared with the original material.
Muller and others (1979) indicated that the problem of precipitation was due to the presence of seed crystals and that if the seed crystals were destroyed, this would result in a stable product. Our results indicate that all the three reasons for precipitation postulated in Section 3.4 are partially responsible although it is not clear how a change in the propor• tions of the a and 3 epimers can produce such a marked change in the stability of calcium glucoheptonate in solution. It is postulated that the
3 epimer hinders growth of the insoluble hydrated a form. The proportions of the a and 8 epimers in the calcium glucoheptonate samples were not altered by autoclaving. This fact reinforces the possible role of seed crystals as a cause of solution instability.
3.4.4 Some comments about USP specifications of calcium glucoheptonate
Only the a form of calcium glucoheptonate is official in the USP
(USP XX, 1980). The stability studies of calcium glucoheptonate solutions
(Table I) indicate that the calcium glucoheptonate supplied by Pfanstiehl which complies with the USP specifications commences to precipitate from solution within a day. It is apparent that if a sample of calcium gluco• heptonate complies with USP specifications (i.e. it consists only of the a form) then it will not be stable in solution. Moreover, the pharmaco• poeia offers no method for the identification of the a form. The USP identification test states that the IR spectrum of the sample under inves• tigation must exhibit maxima only at the same wavelength as a similar preparation of USP Reference Standard of calcium glucoheptonate. It has been found that the IR spectra (KBr pellet) of calcium glucoheptonate - 61 -
Table VII. Proportion of the a epimer in the precipitate
obtained from solutions of calcium glucoheptonate.
Source Proportion of a epimer in Proportion of a epimer in calcium glucoheptonate precipitate (percent) (percent)
Pfanstiehl 100 100
Italsintex 72.4 84.6
Givaudan 71.8 79.9 - 62 -
from different sources (i.e. containing different proportions of the a and
8 epimers) are superimposable. Thus, IR spectroscopy is incapable of distinguishing between the a and 8 epimers of calcium glucoheptonate.
The assay method for calcium glucoheptonate consists of the complexometric estimation of calcium with disodium ethylenediaminetetra- acetate. Hence this method cannot distinguish between the a and 3 epimers.
The rationale behind the choice of the pure a form of calcium glucohepto• nate in the USP is not known. It is apparent that a sample of calcium glucoheptonate, in order to be stable in solution must contain approxi• mately equal proportions of the a and 8 epimers (as in Pfanstiehl (a - 8) mixture). Hence it is suggested that the USP should consider an a - 8 mixture in the monograph for calcium glucoheptonate. Since the relative proportion of the a and 8 epimers is critical for the stability of calcium glucoheptonate solutions, the monograph should include a method for estimating this proportion.
In our studies, based on the close structural similarities of the a and 8 epimers we assumed that the GC response factors of the two epimers would be very close to each other. For the absolute calculation of the relative proportions of a and 8 epimers it would be necessary to have pure reference standards for a-D-glucoheptonic acid y-lactone and
8-D-glucoheptonic acid y-lactone. At present only the a-D-glucoheptonic acid y-lactone is commercially available. - 62a -
PART B
DEVELOPMENT OF ORAL AND PARENTERAL
LIQUID DOSAGE FORMS CONTAINING CALCIUM GLUCOHEPTONATE - 63 -
1. INTRODUCTION
In recent years, solutions of calcium glucoheptonate have shown a
tendency to crystallize on storage (elaborated in Part A, Section 1).
The precipitate formed has been described as lumps of "coral" type
crystals (Chou and Goring, 1979).
Stanley Drug Products Limited, a pharmaceutical organization in
North Vancouver faced a similar problem (Chou and Goring, 1979) which
lead to this collaborative research project to develop stable oral and parenteral solutions of calcium glucoheptonate. - 64 -
2. EXPERIMENTAL
2.1 MATERIALS
*Lactic acid, BDH
*Sodium cyclamate, May & Baker
Sugar, BC sugar
Sodium benzoate, BDH
*Cherry fruit flavor, Givaudan
*Black raspberry flavor, Givaudan
Calcium D-saccharate, ICN Pharmaceuticals
Calcium lactobionate, ICN Pharmaceuticals
* Gift from Stanley Drug Products
The other materials and apparatus used have been described in PartA,
Sections 2.1 and 2.2. - 65 -
2.2 DEVELOPMENT OF ORAL FORMULATIONS
2.2.1 Basic formula
The basic formula (Table VIII) was developed in the Product Develop•
ment Laboratory of Stanley Drug Products Limited. The formulation contains
a mixture of calcium gluconate and calcium glucoheptonate. It has been
found that calcium gluconate acts as a stabilizer for calcium glucohepto•
nate and vice versa when they are used together. It is claimed that a
double salt is formed (Product manual, Pfanstiehl). This combination
permits savings since calcium gluconate is less expensive than calcium
glucoheptonate.
For the oral formulation studies, calcium glucoheptonate from two manufacturers were used:
(i) calcium glucoheptonate USP (Pfanstiehl)
(ii) calcium glucoheptonate (Givaudan)
2.2.2 Use of sugar
The basic formula (Table VIII) contains sodium cyclamate as a
sweetening agent. In order to test the possibility of using sugar as a sweetening agent the basic formula was modified (Table IX). All the oral formulations were broadly divided into two categories based on the sweetening agent used.
2.2.3 Use of stabilizing agent
The stability of solutions containing calcium gluconate may be increased by the addition of a suitable stabilizer such as calcium-D- - 66-
Table VIII. Basic formula
Ingredient Amount
9 13.2 g calcium glucoheptonate calcium gluconate9 11.2 g lactic acid 4.0 mL sodium cyclamate 1.0 g sodium benzoate 0.11 g cherry fruit flavor 0.17 mL black raspberry flavor 0.17 mL water to 100 mL
The final formulation contains 10.79 mg/mL calcium from calcium glucoheptonate and 10.43 mg/mL calcium from calcium gluconate. - 67 -
Table IX. Modified basic formula containing
sugar as the sweetening agent.
Ingredient Amount
calcium glucoheptonate 13.2 g
calcium gluconate 11.2 g
lactic acid 4.0 mL
sugar (granulated) 33.3 g
sodium benzoate 0.11 g
cherry fruit flavor 0.17 mL
black raspberry flavor 0.17 mL
water to 100 mL - 68 -
saccharate and calcium lactobionate (Wade, 1977b). Hence it was decided
to test the effectiveness of these stabilizing agents on the calcium
gluconate - calcium glucoheptonate mixture. When calcium-D-saccharate
or calcium lactobionate was added to the basic formula, a corresponding
amount of calcium gluconate was removed so that the total calcium concen•
tration in the formulation was maintained constant. Formulations were
prepared in which 0, 2, 5 and 10% of calcium gluconate was replaced with
calcium saccharate or calcium lactobionate (Table X).
The use of disodium edetate as a stabilizer of an injectable calcium
gluconate solution has been reported (Welch and Scoratow, 1980). The
effect of this agent on the mixture of calcium gluconate-calcium gluco•
heptonate has also been studied. The details of the formulations are
given in Table X.
2.2.4 Method of preparation of oral formulations
Part A: Calcium gluconate, calcium glucoheptonate and the stabilizing
agent (calcium-D-saccharate,calcium lactobionate or disodium
edetate) were dissolved in 40 mL of boiling water. The heat
was turned off and the solution allowed to cool.
Part B: The sugar, sodium benzoate and lactic acid were dissolved in
25 mL of water with stirring.
After cooling Part A to room temperature, the contents of Part B
were added to Part A and mixed.
Part C: The cherry juice flavor and black raspberry flavor were added
with stirring. The volume was made up to 100 mL and the
solution was filtered. - 69 -
Table X. Oral calcium syrup - formulation details
Basic formula*with sodium Basic formula** with sugar cyclamate (1%-w/y) (33.3% w/v)
calcium D-a , saccharate . 0 0.13 0.32 0.64 0 0.13 0.32 0.64 (percent w/v)
calcium lacto-c bionate 0 0.40 0.98 1.96 0 0.40 0.98 1.96 (percent w/v)
disodium edetate 0 0.10 0.20 0.50 0 0.10 0.20 0.50 (percent w/v)
a equivalent to a replacement of 0,2,5, and 10 percent of calcium gluconate respectively. b Replacement of 2 percent of calcium gluconate with a corresponding amount of calcium D-saccharate so that the total calcium concentration in the formulation was maintained constant. 11.2 g of calcium gluconate is present in 100 mL of the formulation (Table VIII). 2 percent of 11.2 g is 0.224 g. 0.224 g of calcium gluconate contains the same amount of calcium as 0.130 g of calcium D- saccharate. Hence 0.224 g of calcium gluconate was replaced with 0.130 g of calcium D-saccharate. c equivalent to a replacement of 0,2,5 and 10 percent of calcium gluconate respectively.
* Table VIII
Table IX - 70 -
2.3 DEVELOPMENT OF PARENTERAL FORMULATIONS
2.3.1 Basic formula
The USP (USP XX, 1980 ) specifies that calcium glucoheptonate injec• tion must contain between 208 and 233 mg of calcium glucoheptonate per mL.
Hence it was decided to prepare formulations containing 223 mg of calcium glucoheptonate per mL (i.e. 22.3% w/v). The formulation contained no other additives. Two methods of sterilization were used.
(i) autoclaving at 121°C for 20 minutes
(ii) filtration through 0.22 ym membrane filter (Millipore)
For the parenteral formulation development studies, only calcium glucoheptonate USP supplied by Pfanstiehl was used.
2.3.2 Use of stabilizing agents
As in oral formulations, the possible use of calcium D-saccharate, calcium lactobionate and disodium edetate as stabilizing agents was explored. A part of the calcium glucoheptonate was replaced with calcium gluconate and the stability of this combination studied (Table XIII).
2.3.3 Method of preparation of parenteral formulations
Calcium glucoheptonate was dissolved in boiling water. If the formulation contained a stabilizing agent, it was mixed with the calcium glucoheptonate and both solids were dissolved in boiling water, the solution was allowed to cool and then sterilized by autoclaving (at
121°C for 20 minutes) or membrane filtration. - 71 -
3. RESULTS AND DISCUSSION
3.1 STABILITY STUDIES OF ORAL FORMULATIONS
These studies indicate that all the oral formulations commenced
precipitation within 6 months of preparation. However, the use of the
stabilizing agents has decelerated the precipitation reaction, in certain
formulations. The control formulation containing sugar and calcium glucoheptonate USP (Pfanstiehl) (Table XI) commenced to densely precipi•
tate soon after preparation. The use of calcium D-saccharate had a sub• stantial stabilizing effect on this formulation.
Sugar seems to accelerate the precipitation reaction. Most of the formulations (not all) containing sugar commenced precipitation within
3 months. Moreover, dense heavy precipitation was observed only in formulations containing sugar.
Of the three stabilizing agents, calcium D-saccharate seemed to exert slightly greater stabilizing action than calcium lactobionate and disodium edetate.
3.2 STABILITY STUDIES OF PARENTERAL FORMULATIONS
3.2.1 Sterilization by autoclaving
The preliminary stability results (Table XIII) suggest that injectable formulations sterilized by autoclaving do not precipitate.
3.2.2 Sterilization by filtration
Among the various stabilizing agents attempted, only calcium D- saccharate was able to stabilize formulations sterilized by filtration. Table XI. Oral calcium syrup-stability studies
Calcium glucoheptonate USP - Pfanstiehl
Basic formula Basic formula with sugar with sodium cyclamate Formulation details Evidence of precipi tation.(months) 1 3 6 1 3 6
Control +++ ++
2% of calcium gluconate replaced ++ with calcium D-saccharate
5% of calcium gluconate replaced + ++ with calcium D-saccharate
10% of calcium gluconate replaced + - - + with calcium D-saccharate
2% of calcium gluconate replaced +++ ++ with calcium lactobionate
5% of calcium gluconate replaced + + + ++ with calcium lactobionate
10% of calcium gluconate replaced +++ + with calcium lactobionate
0.1% w/v disodium edetate added +++ ++
0.2% w/v disodium edetate added +++ ++
0.5% w/v disodium edetate added +++ - - ++
- clear (no precipitate) + small amount of very fine precipitate at the bottom of the container; precipitation apparent only on shaking the container ++ moderate amount of precipitate +++ dense, heavy precipitate Table XII. Oral calcium syrup-stability studies Calcium glucoheptonate - Givaudan
Basic formula Basic formula with sugar with sodium cyclamate Formulation details Evidence of prec ipitation (months) 1 3 6 ' 1 3 6
Control ++ +
2% of calcium gluconate replaced + + - •-• + with calcium D-saccharate -
5% of calcium gluconate replaced + + - - + with calcium D-saccharate -
10% of calcium gluconate replaced - + + + with calcium D-saccharate
2% of calcium gluconate replaced + + + + with calcium lactobionate
5% of calcium gluconate replaced + + + + with calcium lactobionate
10% of calcium gluconate replaced + + + + with calcium lactobionate
0.1% w/v disodium edetate added + + + +
0.2% w/v disodium edetate added + + + +
0.5% w/v disodium edetate added + + + +
- clear (no precipitate) + small amount of very fine precipitate at the. bottom of the container; precipitation apparent only on shaking the container ++ moderate amount of precipitate +++ dense, heavy precipitate Table XIII. Calcium injection-stability studies
Calcium Glucoheptonate USP - Pfanstiehl
Autoclaved Filtered Formulation details Evidence, of prec ipitation (months) 1 3 6 1 3 6
22.3% w/v solution of calcium +++ glucoheptonate - - -
2.5% of calcium glucoheptonate - - - replaced with calcium D-saccharate - - -
5.0% of calcium glucoheptonate - - - replaced with calcium D-saccharate - - -
2.5% of calcium glucoheptonate - +++ replaced with calcium lactobionate
5.0% of calcium glucoheptonate - - - +++ replaced with calcium lactobionate
25.0% of calcium glucoheptonate _ +++ replaced with calcium gluconate
50.0% of calcium glucoheptonate - - - - replaced with calcium gluconate
0.1% w/v disodium edetate added +++
0.2% w/v disodium edetate added - - - +++
- clear (no precipitate) +++ dense, heavy precipitate - 75 -
Even when as little as 2.5 percent of calcium glucoheptonate was replaced
with calcium saccharate, the formulations have been stable for more than
6 months.
When 25 percent of calcium .glucoheptonate was replaced with calcium
gluconate, the formulation was unstable. However, replacing 50 percent
of calcium glucoheptonate with calcium gluconate resulted in a stable
solution.
Initially calcium glucoheptonate was dissolved in cold water and
then calcium D-saccharate was added to it and this system warmed gently
until complete dissolution of calcium D-saccharate was achieved. However,
it was observed that calcium D-saccharate was unable to exert any
stabilizing action under such conditions. On the other hand, when calcium glucoheptonate and calcium D-saccharate were added to boiling water, the formulations were found to be stable. This suggests that probably a stable complex is formed and this reaction takes place only at high temperature.
The stability studies of the parenteral formulations are continuing.
It is planned to observe the formulations for a total period of two years after their preparation. - 76 -
SUMMARY
1. Only the a epimer of calcium glucoheptonate is official in the USP.
Studies indicate that the pure a epimer is extremely unstable in
solution. On the other hand, a mixture containing nearly equal
proportions of the ct and 6 epimers produces solutions having a
prolonged stability.
2. A gas chromatographic method has been developed for the identification
and estimation of relative proportions of the a and 8 epimers of
calcium glucoheptonate.
3. The instability of calcium glucoheptonate in solutions can be
attributed to:
(i) change from an unstable to a stable form
(ii) presence of seed crystals
(iii) differing proportions of the a and B forms in the calcium
glucoheptonate obtained from various sources.
4. By the use of elemental analysis, X-ray diffraction, IR spectroscopy,
DSC and GC-MS studies, the precipitate obtained from solutions of
calcium glucoheptonate has been identified as a crystalline hydrate
of calcium glucoheptonate.
5. Attempts to develop a stable, oral formulation of calcium gluco•
heptonate have been only partially successful. - 77 -
6. A stable parental formulation of calcium glucoheptonate can be
prepared by autoclaving the final solution. If sterilization by
filtration is desired, then the solution can be stabilized with
calcium D-saccharate or by replacing 50 percent of calcium gluco•
heptonate with calcium gluconate. - 78 -
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