The reason for an increase in decomposition of rifampicin in the presence of isoniazid under acid conditions

Article abstract of DOI:10.1211/146080800128736277

The poor bioavailability of rifampicin from fixed-dose combinations containing isoniazid has been attributed to isoniazid-catalysed degradation under acid conditions in the stomach. The mechanism by which isoniazid enhances rifampicin degradation is not known. The aim of this study was to determine the role of isoniazid in rifampicin decomposition. Degradation studies were performed in 0.1 M HCl at 37°C, in absence and presence of isoniazid. Both rifampicin and isoniazid were analysed. The degradation of rifampicin was increased approximately threefold in the presence of isoniazid. Isoniazid itself was degraded to a lesser extent amounting to one-fifth of the fall of rifampicin. HPLC studies revealed that decomposition of rifampicin in acidic conditions in the absence of isoniazid stopped at the formation of 3-formylrifamycin, while the reaction in the presence of isoniazid proceeded to form a hydrazone between 3-formylrifamycin and isoniazid. The existence of hydrazone was confirmed by its isolation on a preparative column and comparison with an authentic sample synthesized from reaction of 3-formylrifamycin with isoniazid. We suggest that once 3-formylrifamycin is formed, it interacts with isoniazid to form the hydrazone, through a fast second-order reaction. As hydrazones are unstable in acid conditions, 3-formylrifamycin and isoniazid are regenerated in a reversible manner through a slower first-order reaction. In this complex reaction process, rifampicin is further degraded, while isoniazid is recovered.

Full text of DOI:10.1211/146080800128736277

Pharm. Pharmacol. Commun. 2000, 6: 405±410
Received April 4, 2000
Accepted June 6, 2000
# 2000 Pharm. Pharmacol. Commun.
The Reason for an Increase in Decomposition of Rifampicin in
the Presence of Isoniazid under Acid Conditions
SARANJIT SINGH, T. T. MARIAPPAN, NISHI SHARDA, SANJEEV KUMAR AND
ASIT K. CHAKRABORTI
National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar 160 062, India
Abstract
The poor bioavailability of rifampicin from ®xed-dose combinations containing isoniazid
has been attributed to isoniazid-catalysed degradation under acid conditions in the
stomach. The mechanism by which isoniazid enhances rifampicin degradation is not
known. The aim of this study was to determine the role of isoniazid in rifampicin
decomposition.
Degradation studies were performed in 0Á1 M HCl at 37ꢀC, in absence and presence of
isoniazid. Both rifampicin and isoniazid were analysed. The degradation of rifampicin was
increased approximately threefold in the presence of isoniazid. Isoniazid itself was
degraded to a lesser extent amounting to one-®fth of the fall of rifampicin. HPLC studies
revealed that decomposition of rifampicin in acidic conditions in the absence of isoniazid
stopped at the formation of 3-formylrifamycin, while the reaction in the presence of iso-
niazid proceeded to form a hydrazone between 3-formylrifamycin and isoniazid. The
existence of hydrazone was con®rmed by its isolation on a preparative column and
comparison with an authentic sample synthesized from reaction of 3-formylrifamycin with
isoniazid.
We suggest that once 3-formylrifamycin is formed, it interacts with isoniazid to form the
hydrazone, through a fast second-order reaction. As hydrazones are unstable in acid
conditions, 3-formylrifamycin and isoniazid are regenerated in a reversible manner through
a slower ®rst-order reaction. In this complex reaction process, rifampicin is further
degraded, while isoniazid is recovered.
Drug resistance in tuberculosis is due to inap-
propriate prescribing or taking of medications,
effectively resulting in monotherapy. To reduce the
possibility of monotherapy, the World Health
Organization (WHO) and the International Union
Against Tuberculosis and Lung Diseases
(IUATLD) have recommended that antitubercular
drugs should be taken in combination. A number of
combinations of ®rst-line drugs containing rifam-
picin, isoniazid, pyrazinamide, ethambutol, and
streptomycin are now in use. However, the WHO
and IUATLD have cautioned that combinations
containing rifampicin and isoniazid or isoniazid
and pyrazinamide must be of demonstrable bio-
availability. Manufacturers of these combinations
must provide assurance of bioavailability through
appropriate testing. The reasons for the poor
absorption of rifampicin from combination pro-
ducts are not known. Since there are no reactive
groups on rifampicin and pyrazinamide, and only
one on isoniazid, it is unlikely that an interaction is
a cause for the variable bioavailability of rifam-
picin in combination products.
It has been reported that the poor absorption of
rifampicin from combination products may be due
to decomposition of the drug in acidic stomach
conditions, which is accelerated in the presence
of isoniazid (Shishoo et al 1999). However, the
mechanism of this acceleration was not determined.
Jindal et al (1994), studying the development of a
dissolution test method for rifampicin±isoniazid
®xed-dose combinations, indicated that a soluble
complex, presumed to be a Schiff's base between
3-formylrifamycin and isoniazid, was formed
Correspondence: S. Singh, National Institute of Pharmaceu-
tical Education and Research, Sector 67, S.A.S. Nagar 160
062, India.
E-Mail: niper@chd.nic.in

406
SARANJIT SINGH ET AL
during decomposition of rifampicin in acidic con-
ditions, but the identity of the product was not
con®rmed.
protons were con®rmed by the addition of 0Á5 mL
D2O. Chemical shifts are d ppm relative to tetra-
methylsilane. FTIR spectra were recorded in nujol
mull on a Nicolet Impact 410 spectrometer. Mass
spectra were recorded on a Finnigan Mat LCQ
LC-MS.
We established that this product was a hydrazone
formed on reaction of 3-formylrifamycin with iso-
niazid. Based on the reported behaviour of hydra-
zones formed between isoniazid and reducing
sugars (Devani et al 1985), understanding has been
gained on the reasons for the accelerated decom-
position of rifampicin in the presence of isoniazid.
Shishoo et al (1999) used two methods (dual
wavelength UV-vis spectrophotometry and
HPTLC) in their study and focused on the forma-
tion of 3-formylrifamycin from rifampicin in acid
conditions, in the presence and absence of iso-
niazid. The formation of hydrazone was not men-
tioned, as the methods used were not separative to
this product. In a previous study we developed an
HPLC method that was separative to isoniazid,
rifampicin, pyrazinamide, 3-formylrifamycin and
hydrazone (Mariappan et al 2000). Using this
selective method, we determined the extent of
degradation of rifampicin in 0Á1 M HCl at 37ꢀC, in
the presence and absence of isoniazid. Isoniazid
was also analysed.
Degradation studies
For the degradation study on individual drugs,
2Á25 mg rifampicin and 1Á5 mg isoniazid were
separately dissolved in 1 mL 0Á1 M HCl. In another
solution, both drugs were dissolved together. The
solutions were stored at 37ꢀC. Samples were
withdrawn at 50 min, the mean gastric residence
time. As the gastric residence time varies from
15 min to 3 h, additional studies for rifampicin and
isoniazid in combination were performed in which
samples were drawn at 15 min and 3 h. The studies
were carried out in triplicate. The samples were
appropriately diluted and analysed as described by
Mariappan et al (2000).
Isolation of rifampicin±isoniazid interaction
product
Rifampicin (225 mg) and isoniazid (150 mg) were
dissolved in 12 mL methanol and the volume was
made up to 100 mL with water. To 99 mL of this
solution, 1 mL 0Á1 M acetate buffer was added and
the resultant mixture was left overnight at 37ꢀC.
Separation of the product was achieved on a pre-
parative HPLC using methanol±water (45 : 55) as
the mobile phase. The retention times for isoniazid,
the interaction product and rifampicin were 3, 11
and 17 min, respectively. Elutes from 10±15 min
were collected and recycled on the preparative
HPLC until a fraction of suf®cient purity was
obtained. The ®nal fraction was concentrated under
vacuum to obtain the interaction product as a
red solid.
Materials and Methods
Materials
Rifampicin and isoniazid were gift samples from
Lupin Laboratories Ltd (Aurangabad, India) and
were used without further puri®cation. Buffer
materials and all other chemicals were of analytical
grade.
Instrumentation
The analytical HPLC system consisted of a dual
piston reciprocating pump (LC-10ATVP), UV-vis
dual wavelength detector (SPD-10AVP), an auto-
injector (SIL-10ADVP), and an in-line degasser
(DGU-14AM). The data were obtained using
CLASS-VP software (Shimadzu, Kyoto, Japan). A
Supelcosil LC-18-DB column (25 cm 6 4Á6 mm,
5 mm) was used.
Preparative HPLC separation was achieved using
a NovaPrep 200 system (EM Industries Inc., NY)
equipped with a Merck-Hitachi Lachrom L7420
UV-vis spectrophotometer, Hibar RT250-25
column packed with Lichrosorb RP-18 7 mm station-
ary phase and a TurboPrep applications software
(EM Separations Technology, Wake®eld, RI).
13C NMR spectra were recorded on a Bruker
Avance DPX 300 spectrometer. The samples were
dissolved in d6-DMSO. Signals of exchangeable
Preparation of 3-formylrifamycin
3-Formylrifamycin was prepared by hydrolysis of
rifampicin under acid conditions (Gallo & Radaelli
1976). A solution containing 1% w=v rifampicin
and 0Á05% w=v ascorbic acid in 0Á1 M HCl was
stirred at 50ꢀC for 12 h. The resultant suspension
was ®ltered, washed with water and dried. The
structure was con®rmed by 13C NMR which
showed characteristic signals for rifampicin at
170Á69 (C-15), 172Á54 (C-35) and 194Á12 (C-11),
and an additional signal at 196Á61 for the aldehyde
group. No signals were present for CHˆN or for
methyl piperazine carbons. The product was used

ISONIAZID INCREASES DEGRADATION OF RIFAMPICIN
407
in the subsequent reaction without further pur-
i®cation.
was neutralized with dilute ammonia solution
and concentrated in a lyophilizer to afford a solid
product.
Synthesis of hydrazone of 3-formylrifamycin and
isoniazid
Results and Discussion
3-Formylrifamycin (200 mg) was added to 5 mL
0Á1 M HCl and the resulting suspension dissolved
by addition of 8 mL methanol. The solution was
heated at 50ꢀC for 1 h to convert any residual
rifampicin to 3-formylrifamycin. Isoniazid (30 mg)
was added and heating was continued until all of
the isoniazid was consumed (approx. 15 min). The
consumption of isoniazid and the formation of the
product were monitored by HPLC. The solution
Degradation of rifampicin and isoniazid in
0Á1 M HCl
Figure 1 shows the chromatograms at 0 and 50 min
during decomposition of rifampicin, isoniazid,
and a combination of rifampicin and isoniazid in
0Á1 M HCl at 37ꢀC. Comparison of Figure 1A and
B con®rms that only 3-formylrifamycin is formed
Figure 1. Chromatograms recorded at 0 and 50 min, respectively, during decomposition of rifampicin (A, B), isoniazid (C, D) and
rifampicin and isoniazid in combination (E, F) in 0Á1 M HCl at 37ꢀC.

408
SARANJIT SINGH ET AL
Table 2. Loss of rifampicin and isoniazid after storage in
when rifampicin is degraded alone. Figure 1C and
D show that isoniazid is not degraded under the
same conditions as no new peak was detected
(Figure 1D). The areas of the isoniazid peak were
also the same. Figure 1F shows that when rifam-
picin and isoniazid are reacted together, both
rifampicin and isoniazid peak heights decrease and
a new peak emerges along with 3-formylrifamycin.
This new peak was identi®ed as isonicotinyl
hydrazone.
combination in 0Á1 M HCl at 37ꢀC for 15 min and 3 h.
Drugs
Decrease (%)
MeanÆ s.d.
15 min
Rifampicin
6Á65
10Á09
8Á67
0Á49
2Á6
8Á47Æ 1Á73
1Á79Æ 1Á14
Isoniazid
2Á3
3 h
Rifampicin
50Á08
51Á03
48Á91
10Á48
13Á04
7Á45
50Á01Æ 1Á06
10Á32Æ 2Á80
Table 1 shows the extent of the fall in con-
centration of rifampicin and isoniazid at 50 min.
Rifampicin decomposition was 6Á5% in the absence
of isoniazid, while isoniazid alone showed no
decomposition. For rifampicin and isoniazid in
combination, decomposition was 18% and 3Á5%,
respectively. The extent of rifampicin degradation
agrees with Shishoo et al (1999) who reported a
15Á9 and 8Á7% decrease in rifampicin in the pre-
sence and absence of isoniazid, respectively, after
storage at 37ꢀC for 45 min in 0Á1 M HCl.
Isoniazid
Table 3. Spectral data for hydrazone of 3-formylrifamycin
and isoniazid.
Method
Result
The decomposition of rifampicin in the presence
of isoniazid at 15 min was 8Á5%, increasing to 50%
after 3 h. The relative decrease in isoniazid in the
same period was 1Á8 and 10Á3%, respectively
(Table 2).
UV max
FTIR
MS
229 nm, 339 nm and 489 nm
Characteristic band at 1638Á65 cm
1
Molecular ion peak at m=z 843
13C NMR 132Á48 ( CHˆN), 170Á69 (C-15), 172Á54 (C-35),
184.61 ( > C ˆ O), 150.7, 122.6 (pyridine carbons)
These data indicate that decomposition of
rifampicin is accelerated approximately threefold
in the presence of isoniazid. Isoniazid decreases to
a much lesser extent, amounting to approximately
one-®fth of the decomposition of rifampicin.
as a hydrazone between isoniazid and 3-formyl-
rifamycin. The structure was con®rmed by a
molecular ion peak (M-1) at 843 that matched the
molecular weight of hydrazone. The IR spectrum of
the product showed a band at 1638Á65 cm char-
1
acteristic of a CHˆN stretch, a band that was
absent in the spectrum of 3-formylrifamycin. The
13C NMR spectrum of the product showed signals
at 132Á48 ( CHˆN), 170Á69 (C-15), and 172Á54
(C-35) that were characteristic of rifampicin. Sig-
nals due to carbons of the methylpiperazine moiety
at 53Á86 (C-30 and C-50), 50Á14 (C-20 and C-60) and
at 45Á82 (CH3 N) were absent in the spectrum of
the product. New signals at 184Á61 corresponding
to > CˆO and at 150Á7 and 122Á6 for pyridine
carbons provided further evidence of the formation
of the hydrazone.
Characterization of 3-formylrifamycin±isoniazid
interaction product
The spectral data for the isolated product are given
in Table 3. The isolated product was identical
(HPLC, UV, FTIR, MS and 13C NMR) to the
synthesized product. The product was characterized
Table 1. Loss of rifampicin and isoniazid after storage
individually or in combination in 0Á1 M HCl at 37ꢀC for 15 min.
Drugs
Decrease (%)
MeanÆ s.d.
Individual drugs
Rifampicin
5Á40
6Á35
7Á68
0Á01
0Á00
0Á01
6Á48Æ 1Á14
0Á00Æ 0Á01
Suggested mechanism for increased degradation of
rifampicin in the presence of isoniazid
Isoniazid
Rifampicin is degraded in acidic conditions to
generate 3-formylrifamycin and 1-amino-4-methyl-
piperazine through a reversible hydrolysis of the
azomethine ( NˆCH ) bond (Prankerd et al
1992). As shown in Figure 2, the overall position of
the equilibrium is de®ned by the ratio kf=kr, the
forward reaction being pseudo-®rst-order and the
reverse being second-order. The rate of reaction has
Rifampicin ‡ Isoniazid
Rifampicin
19Á65
18Á04
15Á86
3Á61
17Á85Æ 1Á90
3Á52Æ 0Á25
Isoniazid
3Á72
3Á24

ISONIAZID INCREASES DEGRADATION OF RIFAMPICIN
409
been shown to increase in the presence of a trap-
ping agent such as formaldehyde, which reacts
faster with 1-amino-4-methyl-piperazine than 3-
formylrifamycin. In this situation, kf predominates
and the reaction is essentially ®rst-order for more
than three half-lives (Prankerd et al 1992).
Isoniazid is known to decompose at pH 3Á1 to
isonicotinic acid under anaerobic conditions
(Brewer 1977). However, the condensation of
hydrazine group of isoniazid with aldehydes and
ketones to form isonicotinyl hydrazone is of parti-
cular interest in acidic conditions. This reaction is
reported with naturally occurring reducing sugars.
Devani et al (1985) have described the mechanism
which shows that the formation of hydrazone is a
reversible reaction in which the forward reaction is
second-order while the reverse reaction is ®rst-
order (Figure 3).
Figure 3. Pathway for interaction of isoniazid with reducing
sugars.
Based on these observations, and con®rmation
in this study that hydrazone is formed between
explaining the enhanced decomposition of rifam-
picin in the presence of isoniazid under acidic
conditions is suggested in Figure 4. In the presence
of isoniazid, the conversion of rifampicin to 3-
formylrifamycin is expected to primarily follow
®rst-order kinetics instead of a reversible reaction,
as isoniazid acts as a trapping agent for 3-formyl-
rifamycin. The role of isoniazid here may be
equated to that of formaldehyde shown by Prankerd
et al (1992) to trap 1-amino-4-methyl-piperazine
and cause a more rapid fall in rifampicin. The
hydrazone between isoniazid and 3-formyl-
rifamycin is formed by a second-order reaction, as
3-formylrifamycin and isoniazid,
a
pathway
Figure 2. Pathway for decomposition of rifampicin under
acidic conditions.
Figure 4. Proposed pathway for decomposition of rifampicin
in the presence of isoniazid.

410
SARANJIT SINGH ET AL
is the case with hydrazones of isoniazid with
reducing sugars (Devani et al 1985). The hydrazone
is expected to be unstable in acidic conditions so as
to regenerate isoniazid and 3-formylrifamycin by
a pseudo ®rst-order reaction. As the second-order
forward reaction is faster than the preceding
(rifampicin to 3-formylrifamycin) and the follow-
ing (hydrazone to 3-formylrifamycin and isoniazid)
®rst-order reactions, the overall reaction is
favoured towards the formation of hydrazone. As a
result, decomposition of rifampicin to 3-formyl-
rifamycin is pushed forward and an overall increase
in degradation of rifampicin is observed in the
presence of isoniazid.
drug. We established that a hydrazone is formed
between 3-formylrifamycin and isoniazid during
degradation of rifampicin in the presence of iso-
niazid in acidic conditions. The increased decom-
position of rifampicin in the presence of isoniazid
is explained on the basis of a complex consecutive
reaction in which the conversion of rifampicin to
3-formylrifamycin by a ®rst-order reaction is fol-
lowed by a faster second-order reaction leading to
hydrazone. The instability of hydrazone results in
regeneration of isoniazid, thus explaining the small
loss of the latter under the same conditions.
This reaction scheme also explains why only a
small amount of isoniazid is lost during the reaction
compared with rifampicin. This is because iso-
niazid is regenerated due to the reversible nature of
the reaction. Figure 1F substantiates that the fall of
isoniazid is not due to decomposition, but rather to
its involvement in hydrazone formation, as no other
peak due to decomposition of isoniazid was seen in
the chromatogram.
Acknowledgements
We gratefully acknowledge the encouragement of
C. L. Kaul, National Institute of Pharmaceutical
Education and Research, S. A. S. Nagar, India and
the support of K. C. Jindal, Lupin Laboratories Ltd,
Aurangabad, India.
A kinetic study of the reaction suggested in
Figure 4 would be desirable. However, discussion
in the literature indicates that reactions of this type
are too complex for a simple kinetic evaluation.
Connors (1990) indicated that consecutive reac-
tions involving one ®rst-order reaction and one
second-order reaction are kinetically very dif®cult.
Chien (1948) provided closed-form integral solu-
tion to a consecutive irreversible reaction involving
a unimolecular reaction followed by a bimolecular
reaction, but the results are too complex for
straightforward application (Connors 1990). The
present case is still more complicated as instead
of a simple second-order reaction, a bimolecular
reversible reaction is involved in the second step.
Chien (1948) also indicated further complexity
when reversibility is involved.
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This study con®rms that in-vivo degradation of
rifampicin is a major factor contributing to its poor
bioavailability from ®xed-dose combinations con-
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