Transcript Slide 1

Best Practices for OINDP Pharmaceutical Development
Programs Leachables and Extractables
VII. Special Case Compound Classes
PQRI Leachables & Extractables Working Group
PQRI Training Course
September 20-21, 2006
Washington, DC
“Special Cases”
► PAHs - Polyaromatic Hydrocarbons

Also referred to as PNAs (Polynuclear Aromatics)
► N-Nitrosamines
H3C
N
N
O
H3C
► 2-Mercaptobenzothiazole
N
SH
S
PAHs/PNAs as Leachables in OINDP
►
►
►
►
►
►
Historically, the primary source of PNAs is carbon black
which is used as a filler in certain types of rubber (mostly
sulfur cured).
There is some potential for other PNA sources (e.g.
naphthalene contamination).
Some PNAs are known or suspect cancer causing agents
(e.g. benzo(a)pyrene). FDA interest in MDIs traces back to
the late 1980s.
Levels of PNAs in MDIs which employ “black rubber” seals
are typically on the order of ng to low µg/canister.
The FDA historically requires that all elastomers in MDIs be
evaluated and controlled for PNAs.
Analytical methods typically involve GC/MS.
September 2006
PQRI Training Course
3
PNAs Typically Analyzed and Controlled
(EPA Method 610 list)
Naphthalene
► Acenaphthylene
► Acenaphthene
► Fluorene
► Phenanthrene
► Anthracene
► Fluoranthene
► Pyrene
►
September 2006
Benzo(a)anthracene
► Chrysene
► Benzo(b)fluoranthene
► Benzo(k)fluoranthene
► Benzo(e)pyrene
► Benzo(a)pyrene
► Indeno(123-cd)pyrene
► Dibenzo(ah)anthracene
► Benzo(ghi)perylene
►
PQRI Training Course
4
Structures of Some Typical PNAs
naphthalene
phenanthrene
pyrene
benzo(a)pyrene
benzo(ghi)perylene
Trace Organic Analysis
September 2006
PQRI Training Course
6
PNA Analysis in Rubber – Possible Method
► Slice
(or grind) a measured weight of critical
rubber components.
► Add prepared rubber to a boiling flask with a
measured volume of organic solvent (e.g.
toluene).
► Extract via reflux for a pre-optimized time period
(likely 24 hours or greater).
► Remove solvent and reduce in volume.
► Analyze by GC/MS (for example).
 Note that internal standards can be added at various
points in the overall process.
September 2006
PQRI Training Course
7
PNA Analysis in a Suspension Metered Dose
Inhaler Drug Product - Possible Method
►
►
Cool sample MDI canisters (one or several for a composite
sample) over dry ice.
Open canister(s) and filter contents to remove suspended
drug particles.
 Note that filter assembly and catch flask must be cold.
Wash filter contents with organic solvent.
► Evaporate sample to dryness.
► Dissolve residue in a measured quantity of a suitable
organic solvent (e.g. toluene).
► Analyze by GC/MS (for example).
►
 Note that internal standards can be added at various points in the
overall process.
September 2006
PQRI Training Course
8
September 2006
PQRI Training Course
9
A GC/MS System
September 2006
PQRI Training Course
10
Reference
► The
information summarized in the following slides
related to PNA leachables studies is detailed in the
following reference:
Norwood, D.L., Prime, D., Downey, B.P., Creasey, J., Sethi,
S.K., Haywood, P., Analysis of polycyclic aromatic
hydrocarbons in metereds dose inhaler drug
formulations by isotope dilution gas
chromatography/mass spectrometry, Journal of
Pharmaceutical and Biomedical Analysis, 13(3), 293304, 1995.
September 2006
PQRI Training Course
11
GC/MS Analysis of Target PNAs
September 2006
PQRI Training Course
12
EI Spectra of Pyrene
and D10-Pyrene
• Note stability of
the molecular ions
• Note the
characteristic
presence of
doubly-charged
molecular ions
September 2006
PQRI Training Course
13
Benzo(e)pyrene and Benzo(a) pyrene
September 2006
PQRI Training Course
14
Representation Linearity1 and Linearity of Recovery2 Results
for a Drug Product Assay (0.05-2.5µg/inhaler)
Target PNA
Naphthalene
Acenaphthene
Phenanthrene
Fluoranthene
Pyrene
Benzo(e)pyrene
Benzo(a)pyrene
Benzo(ghi)perylene
September 2006
Slope
Intercept
Correlation
0.9731
0.045
0.9998
0.9932
0.033
0.9998
0.658
0.021
0.9998
0.663
0.014
0.9992
1.059
0.067
0.9997
1.061
0.080
0.9990
1.070
0.059
0.9997
1.085
0.069
0.9996
1.034
0.059
0.9998
1.040
0.072
0.9997
1.485
0.056
0.9998
1.487
0.087
0.9994
0.827
0.032
0.9996
0.853
0.054
0.9997
1.320
-0.016
0.9996
1.350
-0.028
0.9998
PQRI Training Course
15
Limit of Detection/Quantitation Results for
Selected Target PNAs
Target PNA
Limit of Detection
(ng/inhaler)
Limit of Quantitation
(ng/inhaler)
Naphthalene
0.7
4
Acenaphthylene
0.7
4
Fluorene
0.9
5
Phenanthrene
0.9
5
Flouranthene
0.7
4
Pyrene
0.7
4
6
30
Benzo(ghi)perylene
September 2006
PQRI Training Course
16
PNA Profile of an MDI Drug Product
September 2006
PQRI Training Course
17
PNAs as Leachables in Metered Dose Inhalers
Target PNA
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Dibenzo(ah)anthracene
Indeno(123-cd)pyrene
Benzo(ghi)perylene
Total
September 2006
Product A
(µg/inhaler)
Product B
(µg/inhaler)
Product C
(µg/inhaler)
0.29
0.43
ND
<0.05
1.96
0.10
1.20
1.26
ND
ND
ND
ND
0.08
<0.05
ND
ND
0.15
0.22
ND
ND
0.88
ND
0.53
0.61
ND
ND
ND
ND
<0.025
ND
ND
ND
0.57
0.45
ND
<0.05
2.14
0.12
1.37
2.13
ND
ND
ND
ND
0.08
<0.05
ND
ND
0.08
0.03
0.06
5.50
2.45
7.02
PQRI Training Course
18
N-Nitrosamines as Leachables in OINDP
►
►
►
►
►
►
Historically, the formation of “nitrosamines” in rubber
involves sulfur curing agents (e.g. thiurams).
The issue of N-nitrosamines in rubber goes back to late
1970s/early 1980s with concern over their presence in
baby bottle rubber nipples. FDA became involved in the
issue. Official analytical methods for rubber developed and
validated.
FDA interest in MDIs (and other OINDP) traces to the early
1990s.
Levels of nitrosamines in MDIs which employ “black
rubber” seals are typically on the order of ng/canister.
The FDA historically requires that all elastomers in MDIs be
evaluated and controlled for nitrosamines.
Analytical methods typically involve GC with “Thermal
Energy Analysis” detection (GC/TEA).
September 2006
PQRI Training Course
19
Target N-nitrosamines
H3C
► N-nitrosodimethylamine
N
H3C
► N-nitrosodiethylamine
O
► N-nitrosodi-n-butylamine
N
N
► N-nitrosomorpholine
O
► N-nitrosopiperidine
► N-nitrosopyrrolidine
O
N
O
N
N
N
N
O
N-nitrosamine Formation
S
S
S
H3C
N
H3C
H3C
C H3
N
H .
N
+
H
C S2
H3C
C H3
H3C
H3C
N
H3C
S.
S
N
C H3
S
heat
H
+
NOX
N
N
O
+
HX
H3C
H3C
X = NO+, N2O3, N2O4, NOZ (Z = Cl, Br, I, thiocyanate)
September 2006
PQRI Training Course
21
N-nitrosamine Analysis in Rubber
(AOAC Method 987.05)
►
►
►
►
►
►
►
►
►
►
Place 5g cut rubber sample in 250mL flask with 100mL methylene
chloride and 100mg propyl gallate, and hold for 17-18h.
Transfer solvent and rubber sample to a Soxhlet extractor.
Spike in internal standard.
Extract for 1 hour.
Add 100mL 5N NaOH and 2g Ba(OH)2 to flask and carefully distill
methylene chloride (discard). Continue distilling 70mL of aqueous
distillate into a sepratory funnel.
Add 300mg anhydrous Na2CO3 to funnel, followed by 50mL methylene
chloride. Extract (repeat twice more). Combine extracts in sepratory
funnel.
Pass through anhydrous Na2SO4 (to dry), into a Kuderna_Danish
apparatus (with appropriate washes).
Concentrate to approximately 4mL.
Remove from KD and further concentrate to 1.0mL with a nitrogen
stream.
Analyze by GC/TEA.
September 2006
PQRI Training Course
22
N-nitrosamine Analysis in Rubber
(AOAC Method 987.05)
Steam distillation
Soxhlet extraction
Extract concentration
Image provided by Rubber Consultants
September 2006
PQRI Training Course
23
Principles of Thermal Energy Analysis
Detection
►
►
►
►
►
N-nitrosamines elute from a GC column into a pyrolyzer,
where they undergo pyrolysis and release nitrosyl radicals
(NO.). The pyrolysis temperature is set low enough so that
nitro-compounds will not pyrolyze.
Nitrosyl radicals are then oxidized with ozone in a reaction
chamber to give electronically excited NO2*.
The NO2* decays back to ground state releasing a photon
at a characteristic wavelength.
This process is known as “chemiluminescence”.
Sensitivity is further increased through use of a filterphotometer for detection.
September 2006
PQRI Training Course
24
6000
0
September 2006
2
4
6
µV
N -dipropylam ine
Internal standard (N D IP A )
N -diethylam ine
N -m ethyletthylam ine
N -dim ethylam ine
10000 12000 14000 16000 18000 20000
8
N -m orpholine
N -pyrrolidine
N -piperidine
N -dibutylam ine
8000
A GC/TEA System – Schematic Diagram
ozone
pyrolysis
vacuum
450oC
cold trap
detector
GC
-130oC
electronics
Time (min)
10
PQRI Training Course
25
A GC/TEA System
Image provided by Rubber Consultants
September 2006
PQRI Training Course
26
A GC/TEA System
Image provided by Cardinal Health
September 2006
PQRI Training Course
27
6000
0
September 2006
2
4
6
PQRI Training Course
14000
16000
N -diethy lam ine
N -m ethy letthy lam ine
N -dim ethy lam ine
12000
N -dipropy lam ine
Internal s tandard (N D IP A )
10000
8
N -m orpholine
N -py rrolidine
N -piperidine
N -dibuty lam ine
8000
µV
18000
20000
GC/TEA N-nitrosamines - Separation
10
Time (min)
28
0
September 2006
2
4
6
PQRI Training Course
8
N -m orpholine
N -py rrolidine
N -piperidine
N -dibuty lam ine
N -dipropy lam ine
N -diethy lam ine
8000
10000
Internal s tandard (N D IP A )
N -m ethy lethy lam ine
N -diethy lam ine
6000
µV
GC/TEA N-nitrosamines - Sensitivity
10 ng/mL
Time (min)
10
29
Some Typical Limit of Detection/Quantitation
Results for Target N-nitrosamines
► AOAC
Method 987.05 LOQs target
acceptance criteria of NMT 10ppb (ng/g) for
an individual N-nitrosamine.
► Based on the LOQs for rubber, MDI methods
should target LOQs around 1 ng/canister.
September 2006
PQRI Training Course
30
N-nitrosamines in OINDP – Points to
Consider
► N-nitrosamines
are usually associated with
sulfur-cured black rubber.
► Even with the sensitivity and selectivity of
the GC/TEA, other peaks are often noted in
OINDP leachables profiles.
► N-nitrosamines are very light sensitive,
which suggests a possible procedure for
identifying “non-nitrosamine” GC/TEA
peaks.
September 2006
PQRI Training Course
31
Analysis of Mercaptobenzothiazole (MBT)
Compounds from Sulfur Cured Rubber by a Liquid
Chromatography – Tandem Mass Spectrometry
(LC-MS-MS) Method
Tianjing Deng*, Shuang Li, Xiaoya Ding and Song
Klapoetke
PPD
8551 Research Way
Middleton, WI 53562
* Corresponding author
September 2006
PQRI Training Course
32
• Mercaptobenzothiazole (MBT) and other benzothiazoles are
common vulcanization accelerators for rubber materials that are
used in pharmaceutical container/systems, such as the gaskets in
the pressurized Metered-Dose Inhaler (pMDI). MBT is of particular
concern since it is considered a potential carcinogen and has been
shown to migrate into drug formulations.
• Due to the toxicological concern and leachability of MBT and other
benzothiazoles, analytical methods have been developed to study
these types of compounds in the fields of food additives and
contaminants (1), contact dermatitis caused by the rubbers (2), as
well as pharmaceutical packaging systems (3). MBT can be
analyzed by gas chromatography (4) but many other
benzothiazoles are thermally-labile and readily decomposed in the
GC inlet. HPLC methods are commonly used to study
September 2006
PQRI Training Course
33
N
N
S
SH
S
S
N
S
S
MBT
MBTS
In this study, a method using liquid chromatography with
tandem mass spectrometer (LC-MS-MS) was developed to
analyze MBT in the sulfur cured rubber. The method is
capable of detecting ng level of MBT in the rubber extracts.
This study demonstrates the feasibility of using detector
with high selectivity, such as LC-MS-MS method, for
extractable/leachable with special toxicological concern that
requires greater sensitivity and specificity.
September 2006
PQRI Training Course
34
RReferences:
1Barnes, K.A., Castle L., Damant, A. P., Read, W. A., and Speck, D. R., Food
Additives and Contaminants, Vol. 20, No. 2, 196-205 (2003).
2Hansson, C., Bergendorff, O., Ezzelarab, M., and Sterner, O., Contact
Dermatitis, 36, 195-200, (1997).
3Gaind, V. S., and Jedrzejczak, K., Journal of Analytical Toxicology, Vol. 17,
34-37, (1993).
4Niessen, W. M. A., McCarney, C. C., Moult, P. E.G., Tjaden, U. R., and Van
der Greef, J., Journal of Chromatography, 647, 107-119, (1993).
5Mathieu, C., Herbreteau, B., Lafosse, M., Morin, Ph., Renaud, M., Cardinet,
C., and Dreux, M., J. High Resol. Chromatogr., 23, (9), 565-566, (2000).
6
September 2006
PQRI Training Course
35
Method Conditions:
HPLC Parameters
Mobile Phase: Water:Methanol:Formic acid
20:80:0.05 (v/v/v)
Flow Rate: 0.2 mL/min
Column: Waters Symmetry C18, 3.5 m, 2 x 100 mm
Column Temperature: 40°C
Autosampler Temperature: Ambient
Injector Volume: 5 L
September 2006
PQRI Training Course
36
“Bench-top” LC/MS Systems
Time-of-flight
Linear ion trap
Triple Quadrupole
September 2006
PQRI Training Course
37
Triple Quadrupole Mass Spectrometer
September 2006
PQRI Training Course
38
MS-MS Spectrum of MBT
+Product (168.0): 64 MCA scans from Sample 1 (direct) of MBT2.wiff
Max. 1.4e7 cps.
168.1
1.4e7
1.3e7
1.2e7
N
135.1
1.1e7
SH
1.0e7
In te n s ity , c p s
S
9.0e6
Mercaptobenzothiazole
(MBT)
8.0e6
7.0e6
124.1
6.0e6
5.0e6
136.2
92.2
4.0e6
109.2
3.0e6
2.0e6
110.2
1.0e6
0.0
65.1
77.1 80.2
30
September 2006
40
50
60
70
80
141.1
95.2 104.1
90
100
110
120
130
m/z, amu
PQRI Training Course
140
169.1
150
160
170
180
190
200
210
220
39
MS-MS Spectrum of MBTS
+Product (333.0): 84 MCA scans from Sample 2 (MBTs) of MBT2.wiff
Max. 5.6e5 cps.
S
167.0
5.5e5
S
N
5.0e5
S
4.5e5
333.2
4.0e5
In te n s ity , c p s
N
S
Dibenzothiazyl
Disulfide (MBTS)
3.5e5
3.0e5
2.5e5
2.0e5
1.5e5
1.0e5
5.0e4
257.2
198.0
0.0
40
September 2006
60
80
100
120
140
160
180
200
220
240
260 280
m/z, amu
PQRI Training Course
300
320
340
360
380
400
420
440
460
480
500
40
Mass Spectrometer:
PE Sciex API 2000/API365 Triple Quadruple Mass
Spectrometer
Io n izatio n M o d e:
D etectio n M o d e:
September 2006
P o sitiv e E lectro S p ray
M RM
M B T @ m /z 1 6 8 /13 5
M B T S @ m /z 3 3 3 /1 6 7
PQRI Training Course
41
Selectivity/Specificity
6 .0 9
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10
11
12
T im e ( m in )
MRM Chromatogram of Extraction Blank:
MBT (blue trace) and MBTS (red trace)
September 2006
PQRI Training Course
42
MRM Chromatograms of MBT (blue) and MBTS (red) in the
500 ng/mL standard solution.
X IC o f + M R M (2 p a irs ): 1 6 8 .0 /1 3 5 .0 a m u fro m S a m p le 5 (S T D -N F ) o f D a ta S E T 2 .w iff
M a x. 5 .7 e 4 c p s .
1 .1 1
5 .7 e 4
5 .5 e 4
MBT
5 .0 e 4
4 .5 e 4
4 .0 e 4
3 .5 e 4
In t
en
s ity
,
cp
s
3 .0 e 4
2 .5 e 4
M BTS
2 .0 e 4
1 .5 e 4
1 .0 e 4
5 0 0 0 .0
0 .0
0 .5
September 2006
1 .0
1 .5
2 .0
2 .5
T im e , m in
PQRI Training Course
3 .0
3 .5
4 .0
4 .5
43
MRM Chromatograms of MBT (blue) and MBTS (red)
in the 30 min TBME Extract.
XIC of +MRM (2 pairs): 168.0/135.0 amu from Sample 22 (30min-1) of DataSET1.wiff
Max. 8.9e4 cps.
1.11
8.9e4
8.5e4
8.0e4
7.5e4
7.0e4
6.5e4
In te n s ity , c p s
6.0e4
5.5e4
5.0e4
4.5e4
4.0e4
3.5e4
3.0e4
2.5e4
2.0e4
1.5e4
1.0e4
5000.0
0.0
September 2006
0.5
1.0
1.5
2.0
2.5
Time, min
PQRI Training Course
3.0
3.5
4.0
4.5
44
Extraction Method
Hansson et al. studied the extraction of MBT/MBTS using different
solvents. They found out that Methyl tert-Butyl Ether (MTBE) is a
good solvent for MBT/MBTS due to its:
• Powerful extraction medium
• Low toxicity
• Inertness to MBT/MBTS
• High volatility
In this study, the rubber was cut into 3 x 3 mm squares. One gram
of the rubber was extracted with 10 mL MTBE for 30 minutes by
sonication. After extraction, the extract was diluted to different
volume using Methanol: Water 50: 50 diluent to give varying MBT
concentrations and filtered using glass fiber syringe filters for LCMS study.
September 2006
PQRI Training Course
45
Extractaed MBT (ppm)
The Extraction Study of MBT by MTBE
from Sulfur-Cured Rubber
200
180
160
140
120
100
80
60
40
20
0
5
10
20
30
40
50
Extraction Time
September 2006
PQRI Training Course
46
Linearity Plot of MBT (50 – 1000 ng/mL)
1200000
Response
1000000
800000
MBT
600000
MBTS
400000
200000
0
0
200
400
600
800
1000
1200
Concentration (ng/mL)
September 2006
PQRI Training Course
47
Repeatability
Calculated MBT Concentration (PPM) in Three Replicates of
Extract.
Calculated Concentration (PPM)
Replicate 1
Replicate 2
Replicate 3
153.4
157.1
149.6
September 2006
PQRI Training Course
Mean
%RSD (n=3)
153.4
2.4
48
LOQ/LOD
The DL of MBT was calculated using S/N ratio = 3.
DL = 6 ng/mL in the solution or 12 pg on column.
MBT
4 .8 3
25
9
24
0
22
0
20
0
18
0
16
0
14
0
12
0
10
0
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
11
12
T im e ( m i n )
MRM Chromatogram of MBT Standard (50 ng/mL)
September 2006
PQRI Training Course
49
Accuracy - Filter Study
A Filter study was conducted to verify that the syringe filter used in
the sample preparation did not reduce the recovery of MBT and
MBTS. Three 500 ng/mL standards were analyzed before and after
the filtration and the area responses of MBT and MBTS were
compared. The percent differences between the filter and nonfiltered samples are less than 2.5% indicating that filtration does not
affect the method accuracy.
C o m po u nd s
B efo re F iltra tio n
A fte r F iltra tion
% D iffe re n ce
September 2006
M e a n a re a re sp o n se s
MBT
MBTS
4 0 82
3 9 90
2 .3
PQRI Training Course
2 1 30
2 1 51
1 .0
50
Accuracy- MBT Recovery
Approximately 360 ng/mL of MBT was spiked into the extract. The sample was
prepared using the sample preparation procedure and analyzed. Three replicates of
spiking samples were prepared and analyzed. The mean recovery of MBT was 87.3%
Recovery Results of MBT
Extract
(ng/mL)
Replicate 1
(ng/mL)
Replicate 2
(ng/mL)
Replicate 3
(ng/mL)
219.5
567.8
552.6
553.2
%RSD
3.5 (n=7)
2.3
2.4
3.0
%Recovery
NA
89.9
86.0
86.1
Calculated
September 2006
PQRI Training Course
51
Summary Points
► The
developed LC/MS/MS method looks
good for MBT, and potentially MBTS.
► MBTS was demonstrated to hydrolyze under
the extraction conditions selected, forming
more MBT.
► This method requires full optimization and
validation.
September 2006
PQRI Training Course
52
Analytical Method Validation
►
System suitability
 Chromatographic parameters (e.g. resolution, tailing)
 Injection precision
►
Precision
 Repeatability
 Intermediate Precision
Selectivity
► Accuracy (three spiking levels)
► Linearity/Range
► LOD/LOQ
► Robustness (e.g. column, mobile phase, temperatures, MS
parameters)
►
September 2006
PQRI Training Course
53
Concluding Points
► “Special
Case” compounds require dedicated
and highly specific analytical methods.
► Dedicated and highly specific analytical
methods have been developed for all
“special case” compounds and compound
classes.
September 2006
PQRI Training Course
54