das arzneibuch und orthogonale methoden - tu … arzneibuch und ... in tooth paste in uk, ......
TRANSCRIPT
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1
Ulrike HolzgrabeChair of the working group
„Arzneimittelsicherheit/Arzneimittelfälschung“of the German Pharmaceutical Society (DPhG)
University of Würzburg
Das Arzneibuch und orthogonale Methoden
- NMR-Spektroskopie -
2
Counterfeit drugs
4 Catagories of counterfeits:• Mislabelled counterfeits
• Counterfeits containing less API• Counterfeits containing the wrong API• Counterfeits containing no API
Ciprobay against anthrax shortlyafter 11th September 2001Tamiflucounterfeits in expectation of a influenza pandemia?May 2007
3
WHO Counterfeits (2006)
poor quality24%
low content21% wrong
packaging5%
wrong API7%
no API43%
Frequency of occurence in different countriesUSA 5 - 7 %GUS > 20 %Africa 50 – 60 %Mexico Latin America 30 % South East AsiaGerman Pharma Health Fund eV.
WHO Estimation
4
The 5th categorydrugs of substandard quality
Active pharmaceutical ingredients (API) consisting of
• New related substances or high amounts of old and/or new related substances
• High amounts of residual solvents• High amounts of heavy metals
Due to changes in synthesis, extraction or purification (~ 25 % of counterfeit drugs)
2
5
Deaths caused by drugs of inaedaquate purity
• 1990 Paracetamol-syrup contaminated with diethylene glycol originated from glycerol (some 80 deaths)
• 1990 Tryptophan-Affair: the change in the production process led to additional impurities causing the Eosinophilia-Myalgia-Syndrome (EMS) some 30 deaths
• 2000 Gentamicin in high concentrations (off-label-use) caused the death of some 60 people in the USA
• 2007/8 Heparin with anaphylactoid, oversulfatedchondroitin sulfate, approx. 100 deaths in USA
• 2006-9 Diethylene glycol in glycerin from China: dozens of deaths in Panama, in tooth paste in UK, in syrup for teething children in Nigeria
6
Shift of production plants
In 1990, ~80 % of the APIs wereproduced in Europa & USA In 2009, ~80 % of
the APIs wereproduced inChina & Indien
Problem: low number of inspections in China!
7
The EDQM has conduced160 API inspections in China and India in the 10 years:
More than 50 certificatesof suitability weresuspended or withdrawn
8
The situation is not as good as claimed by the EDQM.
• Not all monographs are as modern as they should be.
• There are ways to circumvent the purity control = non detected impurities
Orthogonal methods are necessary!
qNMR, Raman, IR spectroscopy, other separation techniques than HPLC, e. g. capillary electrophoresis
3
9
Outline
• Fundamentals
• The gentamicin case
• Diethylene glycol in glycerin
• qNMR of Heparin: OSCS, dermatan and more
• Conclusion
10
Outline
• Fundamentals
• The gentamicin case
• Diethylene glycol in glycerin
• qNMR of Heparin: OSCS, dermatan and more
• Conclusion
11
NMR is a Primary MethodComité consultatif pour la quantité de matière (CCQM)Definition April 2000:A primary method of measurement is a method having the highest metrological properties, whose operation can be completely described and understood, for which a complete uncertainty statement can be written down in terms of SI units• A primary direct method measures the value of an
unknown without reference to a standard of the same quality
• A primary ratio method measures the value of a ratio of an unknown to a standard of the same quantity; its operation must be completely described by a measurement equation.
B. King, Accredit. Qual. Assur. 5 (2000) 266-271 & 429-43612
NMR spectroscopy in quantitative analysis
NcI S
The intensity of a NMR-signal is given by the area under the specific signal. The intensity I of a signal is directly proportional to the number of nuclei N evoking that signal. The relation between the signal intensity I and the number of observed nuclei is given by:
Integration from - until +
X
Y
Y
X
Y
X
NN
II
nn Relative method:
molar ratio
Absolute method:
Main component Px
Std
Std
Std
X
X
Std
Std
XX P
mm
MM
NN
II
P
4
13
Factors to be consideredPrecision of the integrals determines the accuracy of quantification
• depends on the noise level of the spectrum
• depends on the line shape
• quality of shimming
• choice of the window function
• phase-, baseline- and drift corrections (user!)
• relaxation: determination of T1 of the signals considered!
• integral interval (64 times the full width at half signal height)
Optimization and validation of the method is necessaryDiehl, Malz, Holzgrabe, Spectroscopy Europe 17 (2007) 5: 15 – 19; Holzgrabe: Progr. NMR spectroscpy 57 (2010) 229-240 14
Problems to Overcome
• Sensitivity- high field strength, - invers and (micro) cryo probes, - the higher number of scans (square roots on N!) the higher the
S/N ratios: > 250:1 for 1H, > 300:1 for 19F and > 600:1 for 31P are necessary for sdv < 1%,
- gradient shimming (quality)
• Signal overlap (signal purity) - choice of the solvent (e.g. ASIS), - different sample concentrations, - pH value of the solution, if analytes are basic or acid, - addition of auxiliary reagents like cyclodextrins or
lanthanide shift reagents, - temperature, which also affects the signal separation
• Rotational side bands & 13C satellites
)/(/ NSnNS n )/(/ NSnNS n
15
ApplicationsNMR spectroscopy can be used
• to identify a drug or an excipient
• to evaluate the level of impurities (and to elucidate the structure)
• to observe the course of decomposition
• to evaluate residual solvents
• to determine the isomeric composition:
the ratio of diastereomersthe ee by means of chiral additive
• to assess a single drug or drug composition
• to characterize a polymer
• To identify the counterion (organic)
• to unravel counterfeit drugs (e. g. 1H, 13 C, 19 F NMR or DOSY, NOESY, etc.)
16
NMR in International Pharmacopoeiae
Identity: DAB 9: Gentamicin (1H)PhEur 6: Buserelin (1H), Goserelin (13C), Tobramycin (1H)
Heparins low-molecular-mass, unfractionated Heparin (1H), Vaccines: HaemophilusType b conjugate vaccine, Meningococcal Group C conjugate vaccine, Pneumococcal polysaccharide conjugate vaccineSalmon oil farmed (13C)
BP 1998: Hydrocortisone Sodium Phosphate (1H) USP 26: Amylnitrite isomers (1H)
Tests: DAB 9: GentamicinPhEur 6: Poloxamer: ratio of oxypropylene/oxyethylene,
Hydroxypropylbetadex: molar substitution,Lauromacrogol 400: average chain length of fatty alcohol
and ethylene oxide (in preparation)USP 30: Orphenadrine citrate: meta/para isomer
Assay: USP 30: Amylnitrite isomers
5
17
Outline
• Fundamentals
• The gentamicin case
• Diethylene glycol in glycerin
• qNMR of Heparin: OSCS, dermatan and more
• Conclusion
18
Gentamicin case
19
Gentamicin case
R1 R2 R3Gentamicin C1 -CH3 -H -CH3
Gentamicin C1a -H -H -HGentamicin C2 -CH3 -H -HGentamicin C2a -H -CH3 -HGentamicin C2b -H -H -CH3
R4 Sisomicin -H
Netilmicin -CH2-CH3 (Pos. 30/31)
O
O
NH2OH
O NH2NH2
OOH
CH3 NHOH
CH3
NH
R1 R2
R3
garosamine
2-desoxystreptamine
purpurosamine
O
O
NH2
OH
O NH NH2
OOH
CH3 NHOH
CH3
R4
NH2
garosamine
2-desoxystreptamine
4,5-dehydropurpurosamine C
OH
OH
O NH2NH2
OOH
CH3 NO
CH3
OH
OH
OHNH2
NH2
garamine 2-desoxystreptamine
PhEur 6.0: Gentamicin sulfate is a mixture of the sulfates of antimicrobial substances produced by Micromonospora purpurea,the main compounds being gentamicin C1, C1a, C2, C2a, and C2b.
20
ECD: Gentamicin (G22)
C1
C1a
40.62
C2/C2aC2b
Siso
100
105
110
115
120
125
0 10 20 30 40 50 60 70
Zeit (min)
Inte
nsitä
tG22
C1C1aC2/C2aGADSA
ParoC2bSisoG A
D SA
Par o
100
102
104
106
108
110
112
0 2 4 6 8 10
( G 2 2 )
ECD: Gentamicin (G22)
100
102
104
106
108
110
0 10 20 30 40 50 60 70Zeit (min)
Inte
nsitä
t
C1
C1a
C2a
C2
Siso
mic
in
HPLC, pulsed amperometric detection
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21
Comparison PhEur HPLC & the CE method
Adams et al. JPBA 18 (1998) 689 Kaale et al. J. Chromatogr. A 895 (2000) 6722
Aiming for more selectivity: MEKC
Wienen, Holzgrabe, Electrophoresis, 24 (2003) 2948Validation: Kühn, Weber, Holzgrabe, JPBA 48 (2008) 612
MEKC
Typical impure sample, containing much sisomicin
Wienen, Deubner, Holzgrabe, Pharmeuropa15 (2003) 273
23
DAB 9 - NMR an orthogonal method
Identity
Tests
60 MHz NMR spectrometer!
D.H. Calam et al. J. Pharm. Pharmacol. 30 (1978) 220 24
O
O
NH2NH
R3
R1R2
OH
O NH2NH2
OOH
CH3 NHOHCH3
R1 = -H, -CH3R2 = -H, -CH3R3 = -H, -CH3
1.52.02.53.03.54.04.55.05.56.0 ppm
anomeric
anomeric
H-2ab
2
H-4ab‘s
4
H-2ab‘s
2
H-3d
3
Side chain
N-CH3
N-CH3
H-3
3
H-5
5
CH3
CH3
HDO
15mg GM in 650 µl D2O
Sample 31
400 MHz-Spectrometer
2.75; 295
1.25; 1.35
7
25
Sample No. 1 Sample No. 9
Sample No. 19 Sample No. 35
5.15.25.35.45.55.65.75.85.96.0 ppm5.15.25.35.45.55.65.75.85.96.0 ppm
5.15.25.35.45.55.65.75.85.96.0 ppm 5.15.25.35.45.55.65.75.85.96.0 ppm
? ?
Signals of Anomeric hydrogens
?Purposamine Signals
Garosamine signals
26
Assignments of the signals
5.05.15.25.35.45.55.65.75.85.9 ppm
C2a C1 C2 C1a
netilmicin,sisomicin (H-20)
netilmicin,sisomicin (H-23)
garosamine part (H-1)
??
purpurosamine part (H-20)
JI-20B
garamine
R. Deubner, F. Wienen, U. Holzgrabe, Magn. Res. Chem. 41(2003) 589 R. Deubner, U. Holzgrabe, J. Pharm. Biomed. Anal. 35 (2004) 459-467
ONH2 NH2
HH
R
ONH2 NHCH3
CH3
H
R
ONH2 NH2
CH3
H
R
O
R
NH2 NH2
HCH3 O
R
NH2 NHCH3
HH
Gentamicin C1aGentamicin C1 Gentamicin C2 Gentamicin C2a Gentamicin 2b
27
Comparison NMR-MEKC
5.05.15.25.35.45.55.65.75.85.96.05.05.15.25.35.45.55.65.75.85.96.0
C2a/C1/C2/C1a
JI-20B ?Sisomicin
GA
pure sample dirty sample
28
3D score plot of a PCA model
High puritysamples
Average puritysamples
Low puritysamples
Atypicalgroup
Results of PCA: High portions of gentamicin C2 is generally indicative of low amounts of impurities.
Winter, Deubner, Holzgrabe, J. Pharm. Biomed. Anal. 38 (2005) 833-839
8
29
Conclusions - GentamicinNMR spectroscopy can be used in impurity profiling!
Gentamicin profiling:
• 8 different composition pattern could be identified• Some patterns occurred in samples of different origin• Samples from same sources exhibited different
patterns indicating different producers (than known to the MAA holder and known to the authorities)
• “Dirty” samples were found to be the samples which were responsible for deaths and side effects
References: F. Wienen, R. Deubner, U. Holzgrabe, Pharmeuropa 15 (2003) 273 R. Deubner, F. Wienen, Magn. Res. Chem. 41 (2003) 589F. Wienen, U. Holzgrabe, Electrophoresis, 24 (2003) 2948R. Deubner, U. Holzgrabe, J. Pharm. Biomed. Anal. 35 (2004) 459 U. Holzgrabe, R. Deubner, F. Wienen, American Pharmaceutical Outsourcing 5 (2004) 24 etc.
30
Outline
• Fundamentals
• The gentamicin case
• Diethylene glycol in glycerin
• qNMR of Heparin: OSCS, dermatan and more
• Conclusion
31
• 1985 Diethylene glycol in Austrian Wine
• 1990 Paracetamol-syrup contaminated with diethylene glycol originated from glycerol (some 80 deaths)
• 2006 Diethylene glycol in glycerin from China: dozens of deaths in Panama
• 2007 Diethylene glycol in glycerin found in tooth paste in UK
• 2009 Diethylene glycol in glycerin in syrup for teething children in Nigeria
Diethylene glycol in glycerin
32
Diethylene glycol in glycerin
Every impurity 0.1 %Total of all 0.5 %
Ph. Eur. 6.3
9
33
Diethylene glycol in glycerinPurity control of glycerin bymeans of 1H NMR spectroscopy
1H NMR spectrum of glycerin contaminated with 4 % DEG, EG and PG (400 MHz, pyridine-d5, 128 scans).
EG
DEG DEGPG
PG
G
G
4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 1.4 ppm
Limit of related substances in PhEur 6.0
• DEG ≤ 0.1 % (GC)
1H NMR spectroscopy
• quantitative analysis of DEG, EG and PG
• LOQ << 0.1 %
• analysis time ~ 30 min
34
Diethylene glycol in glycerinPurity control of glycerin by meansof 13C NMR spectroscopy
13C NMR spectrum of glycerin contaminated with DEG, EG and PG (400 MHz, pyridine-d5, 512 scans).
G
EG
DEGDEG
PG
G
74 73 72 71 70 69 68 67 66 65 64 63 62 ppm
74.4 74.0 73.6 ppm
G
1% DEG
65.0 64.5 ppm
1% EG
G
* *
* *JC,C
JC,C
13C NMR spectroscopy
• LOQ ~ 0.1 %
• increased analysis time
Beyer, Holzgrabe, in preparation
35
Outline
• Fundamentals
• The gentamicin case
• Diethylene glycol in glycerin
• qNMR of Heparin: OSCS, dermatan and more
• Conclusion
36
Heparin Case
The History• In 2008, some 900 cases of adverse events
associated with the use of heparin were reported to the FDA and the BfArM, some 200 died.
• The adverse events could be assigned to the occurrence of oversulfated chondroitin sulfate in heparin, originated from China (mostly Baxter)
• In the first stage, an NMR and CZE method were developed in order to limit this impurity.
• In the second stage, the NMR method went into the identification section of USP and PhEur, a new anion-exchange HPLC and a new biological assay were introduced in the test section, etc.
10
37
Heparin Case
„oversulfated“ Chondroitin sulfateGalactosamine + Glucuronic acidR1-R4 = sulfatedOSCS
Dermatan sulfateGalactosamine + Iduronic acid
HeparinGlucosamine + Iduronic acid,N-acetylated Glucosamine + Glucuronic acid
O
CH2OSO3Na
NHSO3Na
OH
O
OSO3Na
OHCO2Na O O
n
)
O
CH2OR4
NHCOCH3
O
O
OSO3R2
OR1
CO2Na
n
R3
OO
O
CH2OH
NHCOCH3
O
O
OSO3Na
OHCO2Na
n
NaO3S
OO
)
O
CH2OR'
NHCOCH3
O
O
OH
CO2Na
OH n
R
OO
Chondroitin sulfate A/C Galactosamine + Glucuronic acidR = R´ = sulfated
38
1.52.02.53.03.54.04.55.05.56.06.5 ppm
D2O
CH3
MS
pure Heparin, D2O, 400 MHz, 315K
Heparin - 1H NMR
IS: malic acid
39
Test on IdentityHeparinChondroitin Sulfate A and CDermatan SulfateOSCS (A/C)OSDSHyaluronic acidDetermination of Animal Source at 353 K
Amount Quantification by internal standardby-products Dermatan Sulfate LOD approx. 0.5%Contamination OSCS LOD approx. 0.1%Solvent residue Methanol, Ethanol, DMF LOD approx. 50 ppm dir.Drug Products By water suppressionLMW Heparine Enoxiparin, Fraxiparin, etc
What can be done by means of NMR spectroscopy?
Heparin Cases
40
Heparin 1H NMR spectrum
1.52.02.53.03.54.04.55.05.56.08.5 ppm
FA
MeOH
EtOH
NaOAcacetone
2.02.1ppm
Hep
DSOSCS
N-acetyl region
EtOH
11
41(ppm)
1.61.71.81.92.02.12.22.32.4
Chondroitin sulfate A/C
OSCS
Dermatan = Chondroitin sulfate B
Unfractionated Heparin
Contaminated Heparin = OSCS
Heparin - 1H NMR
42
Heparin - 1H NMR
pH variation?
pH variations and content of Ca2+ and Mn2+
43
Heparin – LOD NMR
1) pure Heparin spiked with OSCS2) pure Heparin spiked with
contaminated Heparin of known content
Experimental parameters• 400 MHz• 90°-Puls (versus 30°)• NS = 128 • T1 (CH3) = 1,44s → 5 x T1• T = 315K (versus 353K)• Mt = ~ 18min KM
H
2.002.052.102.152.202.252.302.352.40 ppm
~ 0,9% OSCS~ 0,4% OSCS~ 0,2% OSCS~ 0,1% OSCS
13C-Sat. 13C-Sat.
LOD = 0.1 % !
T. Beyer, B. Diehl, G. Randel, E. Humpfer, H. Schäfer, M. Spraul, C. Schollmayer, U. Holzgrabe, J. Pharm. Biomed. Anal. 48 (2008) 13-9, and Pharmeuropa Bio 2008-1, 31-39
OSCS
44
Heparin
0
5
10
15
20
25
30
0 20 40 60 80 100 120 140
OSC
S [%
]
heparin batch
Analysis of ~ 150 batches in Germanycontent of OSCS
Beyer, Matz, Brinz, Rädler, Wolf, Norwig, Baumann, Alban, Holzgrabe,Eur. J. Pharm. Sci. 40 (2010) 297-304
12
45(ppm) 4.44.64.85.05.25.45.6
DermatanSulfate
(ppm) 4.44.64.85.05.25.45.6 (ppm) 4.44.64.85.05.25.45.6 (ppm) 4.44.64.85.05.25.45.6
(ppm) 2.002.022.042.082.102.122.14
Dermatan Sulfate Heparin
(ppm) 2.002.022.042.082.102.122.14 (ppm) 2.002.022.042.082.102.122.14 (ppm) 2.002.022.042.082.102.122.14
DermatanSulfate
Heparin
Evaluation of Dermatan sulfateStandard Addition of Dermatan Sulfate to Hepariny = 1,0832x + 4,4586
R2 = 0,9956
0,0
2,0
4,0
6,0
8,0
10,0
12,0
14,0
16,0
18,0
0,0 2,0 4,0 6,0 8,0 10,0 12,0
Added Dermatan Sulfate [%]
Inte
nsity
Ace
tyl S
igna
l
Standard addition of DS to heparin
T. Beyer, B. Diehl, G. Randel, E. Humpfer, H. Schäfer, M. Spraul, C. Schollmayer, U. Holzgrabe, J. Pharm. Biomed. Anal. 48 (2008) 13-9
46
Heparin
Analysis of ~ 150 batches in Germanycontent of dermatan sulfate
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100 120 140
DS
[%]
heparin batch
Beyer, Matz, Brinz, Rädler, Wolf, Norwig, Baumann, Alban, Holzgrabe,Eur. J. Pharm. Sci. 40 (2010) 297-304
47
Heparin – PCA analysis
Analysis of ~ 150 batches in GermanyScore plot of PCA
(presence (> 1%) of OSCS +1, absence of OSCS -1)
-2
-1
0
1
2
-2.0 -1.5 -1.0 -0.5 0 0.5 1.0 1.5 2.0 PCA_qualitative…, X-expl: 13%,11%
-1.000
-1.000-1.000
1.000
1.000
-1.000
-1.000
-1.000
-1.000
-1.000-1.000-1.000
1.000
-1.000-1.000-1.000
-1.000
1.000
1.000-1.000-1.000
-1.000
1.0001.000
-1.000-1.000
1.000
-1.000
-1.000-1.000-1.000
-1.000
-1.000
-1.000
1.000
-1.000
-1.000 1.0001.0001.000
-1.000
1.000
-1.000-1.000
-1.000
-1.000
-1.000
-1.000
-1.000
-1.000
-1.000
-1.000
-1.000-1.000
-1.000-1.000-1.000
-1.000-1.000
-1.000
-1.000-1.000
1.000
1.000
-1.000
-1.000
1.000
-1.000
1.000
-1.000
-1.000 -1.000
-1.000
-1.000
-1.000
-1.000-1.000
1.000-1.000
1.000
-1.000
-1.000
1.000
-1.000
1.000
-1.000
-1.000
-1.000 1.000
-1.000
-1.000
-1.000
-1.000-1.000
-1.000
1.000
-1.000-1.000-1.000
-1.000
1.000
-1.000
-1.000-1.000
1.000
-1.000-1.000-1.000-1.000-1.000
1.0001.000
-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000
-1.000
-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000-1.000
-1.000
-1.000
-1.000
-1.000
PC3
PC4 Scores
Beyer, Matz, Brinz, Rädler, Wolf, Norwig, Baumann, Alban, Holzgrabe,Eur. J. Pharm. Sci. 40 (2010) 297-304
48
Heparin - 1H NMR - PCA
OSCS
DS
Heparin N-acetyl
Increasing OSCS
Increasing DS
Prior to bucketing spectra were aligned and scaled to the N-acetyl signal of heparin at 2.05ppm. The PC1 and PC2 scores represent 83.6% and 12.6% of the total variance in the bucket table, respectively. The PC1 scores are dominated by the effect of OSCS contamination whereas PC2 variation results from DS concentration variation.
13
49
Heparin - Residual Solvents
1.52.02.53.03.54.04.55.05.56.08.5 ppm
FA
MeOH
EtOH
NaOAcacetone
2.02.1ppm
Hep
DSOSCS
N-acetyl region
EtOH
50
Heparin
94
53
17
511
ethanol sodium acetate acetone methanol formic acid
Analysis of ~ 150 batches in Germanycontent of residual solvents
No. batches
Up to 10 %
Traces
Beyer, Matz, Brinz, Rädler, Wolf, Norwig, Baumann, Alban, Holzgrabe,Eur. J. Pharm. Sci. 40 (2010) 297-304
51
Heparin – PCA analysis
Analysis of ~ 150 batches in GermanyCorrelation between all residual solvents, OSCS, DS
Loading plot
-1.0
-0.5
0
0.5
1.0
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8PCA_qualitative…,X-expl: 13%,11%
DS
OSCS
Acetate
EtOH
MeOHAcetoneFormic Acid
Peak 1.35 ppm
PC3
PC4 X-loadings
Beyer, Matz, Brinz, Rädler, Wolf, Norwig, Baumann, Alban, Holzgrabe,Eur. J. Pharm. Sci. 40 (2010) 297-304
52
Heparin – bovine versus porcine
Beyer, Diehl, Holzgrabe, Bioanalytical Rev., in press
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 ppm
porcine
bovine
N-C
H3
NaO
AcN-acetylsignal size
14
53
Heparin – Calcium versus Sodium
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 ppm
Hep-Ca2+
Hep-Na+
Glu
-H1
Id-H
1
Id-H
5
Glu
-H1
Id-H
1
Id-H
5
N-C
H3
Beyer, Diehl, Holzgrabe, Bioanalytical Rev., in press
2.3 2.2 2.1 2.0 ppm
Hep
OSCS
Hep-Ca2+
Hep-Na+
54
Heparin – PCA analysis
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
4
3
21
HOD
← T
SP
H1
Glc
NA
c/G
lcN
S, 6
SH
1 Id
oA2S
H2
Glc
NS
met
hylG
lcN
Ac
USP/PhEur: Identification test by means of NMR limits OSCS, DS and residual solvents
Blue regions empty
Typical for porcineheparin
55
Heparin
Analysis of ~ 150 batches in GermanyOptical rotation (> 35o)
PhEur 6.0
10
20
30
40
50
60
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
spec
ific
rota
tion
[º]
heparin batch
Heparin Heparin + DS Heparin + OSCS Heparin + DS + OSCS
20
25
30
35
40
45
50
55
0,0 5,0 10,0 15,0 20,0 25,0 30,0
spec
ific
rota
tion
[º]
OSCS + DS [%]
Beyer, Matz, Brinz, Rädler, Wolf, Norwig, Baumann, Alban, Holzgrabe,Eur. J. Pharm. Sci. 40 (2010) 297-304
56
Heparin – PLS1 analysis
-0.10
-0.05
0
0.05
0.10
-0.20 -0.15 -0.10 -0.05 0 0.05 0.10 0.15 PLS1_1st, X-expl: 10%,6% Y-expl: 8%,59%
-1.000 -0.600 -0.200 0.200 0.600 1.000
DS_ET
nv_AC
nv_AC
OSCS_
OSCS
nv_ET
DSDS_ET
DSDS_ET
DS_ET
DS_ET
OSCS_
DS_ET
DS_ET
nv_ET
nv_ET
OSCS_
OSCS_
nv_AC
DS_ACnv_AC
OSCS_
OSCS_
nv_AC
nv_AC
OSCS_
DS_AC
DS_AC
DS_ACDS_ACnv_ACnv_AC
nv_AC
OSCS_
DS_AC
nv_AC
OSCS_ OSCS_OSCS_
DS_AC
OSCS_
nv
nv
nv
nv_AC
nv_ET
DS_ACnv_AC
nv_AC
DS_ET
nv_ET
DS_ET DS_ET
DS nv_AC
nv_ET
nv
PC3
PC1 Scores
Raman data; +1 OSCS contaminated; +1 pure heparin
Norwig, Beyer, Brinz, Holzgrabe, Diller, Manns, Pharmeuropa Sci. Notes. 2009-1, 17-24.
15
57
Heparin – PCA analysis
Analysis of ~ 150 batches in GermanyBiological activity (sheep plasma clotting assay)
0
50
100
150
200
250
0 20 40 60 80 100 120 140
biol
ogic
al a
ctiv
ity [I
U/m
g]
heparin batch
All samples fulfilled the requirements of the PhEur 6.0
Beyer, Matz, Brinz, Rädler, Wolf, Norwig, Baumann, Alban, Holzgrabe,Eur. J. Pharm. Sci. 40 (2010) 297-304
58
Heparin – PCA analysis
Analysis of ~ 150 batches in GermanySheep versus human plasma clotting assay
Alban, Lühn, Schiemann, Beyer, Norwig, Schilling, Rädler, Wolf, Matz, Holzgrabe,Anal. Bioanal. Chem. In press
OSCS 5.5-10% >10%DS <1.8% 1.8% <1.8% 1.8% <1.8% 1.8%
non-acceptably contamined heparin samples
100
120
140
160
180
200
220
Pote
ncy
of H
epar
in (I
U/m
g)
sheep plasma-based aPTT 180 IU/mg < 180 IU/mg human plasma-based aPTT 180 IU/mg < 180 IU/mg
0
20
40
60
80
100
120
0 20 40 60 80 100OSCS content (%)
Rel
ativ
e Po
tenc
y of H
epar
in (%
)
sheep plasma-based aPTT human plasma-based aPTT
59
Outline
• Fundamentals
• The gentamicin case
• Diethylene glycol in glycerin
• qNMR of Heparin: OSCS, dermatan and more
• Conclusion
60
Advantages of NMR spectroscopy
• Integration of signals is more precise and accurate than HPLC analysis
• Often, no isolation of the impurity necessary
• (No) expensive chemical reference substances necessary
• Additional structural information of impurities, isomers etc.
• NMR can be quicker (no equilibration time as with HPLC)), easy to perform and more specific high reproducibility
16
61
qNMR as an orthogonal method
Using NMR spectroscopy as an additional orthogonal method (to e. g. HPLC) because
• is not optimized for one synthesis pathway
• it cannot be manipulated
• gives normally more than one signal for an additional component/impurity
• deviations from a typical signature of a drug can be easily detected by simple inspection (small molecules) or statistical methods (e.g. PCA, PLS) in the case of „biologicals“
• difficult to hide an impurity
62
Further reading: HolzgrabeProgr. NMR spectroscopy, 57 (2010) 229-240
HolzgrabeEncyclopedia of Spectroscopy and Spectrometry, Eds. J. Lindon; G. Traner, D. Koppendahl, 2nd Ed. AP 2010, Vol. 3, p. 2331-233
Holzgrabe, BeyerBioanalytical Reviews, in press
63
Acknowledgement
• Coworkers: F. Wienen, R. Deubner, C. Schollmayer & T. Beyer
• Bruker Spectrospin: Humpfer, Spraul, Schäfer
• Federal Institute for Drugs and Medical Devices for providing samples of gentamicin and heparin, and financial support
• Federal Ministry of Economics and Technology (BMWI), the German Federation of Industrial Cooperative Research Associations "Otto von Guericke" (AiF) for financial support
Thank you for attention!