2021 Labs 1,2,3,4 - Students

EXPERIMENT #1:
A. ASSAY: % Label Claim of Acetaminophen (500 mg) and Caffeine (65 mg) in tablets
B. WEIGHT VARIATION of Acetaminophen (500 mg) and Caffeine (65 mg) in tablets
SOLVENT MIXTURE:
Prepare ~ 900 mL of a mixture of methanol and glacial acetic acid in the ratio 95:5.
INTERNAL STANDARD SOLUTION:

Weigh ~300.0 mg benzoic acid into 50 mL volumetric flask and add ~ 30 - 40 mL methanol.
Sonicate for 1 minute with periodic shaking to dissolve. Dilute to volume with methanol. Mix.
STANDARD STOCK SOLUTION:
Weigh 250.0 mg of acetaminophen reference standard and 32.5 mg of caffeine reference standard into a
250 mL volumetric flask. Add 75 mL of Solvent Mixture and shake (or/and sonicate) until the standards
dissolve. Add Solvent Mixture to volume. Mix!
WORKING STANDARD PREPARATION:

Transfer 5.0 mL of the Standard Stock Solution and 3.0 mL of the Internal Standard Solution into a 50 mL
vol. flask and dilute to volume with Solvent Mixture. Mix! This solution contains 0.1 mg/mL of
acetaminophen reference standard and 0.013 mg/mL of caffeine reference standard.
ASSAY (I.E., SAMPLE) PREPARATION:

Individually weigh 10 tablets or caplets and record both the individual weights and the average
tablet weight (ATW). You will need these weights for your Weight Variation calculations.
Next crush 5 tablets and mix the powder using a mortar and pestle. (Note: USP requires 20, but
we do not wish to discard large amounts of unused powder)
Weigh an amount of tablet powder equivalent to 250 mg acetaminophen. Transfer into a 100 mL
vol. flask. Add 75 mL of Solvent Mixture and shake mechanically for 30 minutes. Dilute with
Solvent Mixture to volume. Mix!
Transfer 2.0 mL of this solution and 3.0 mL of the Internal Standard Solution into a 50 mL vol.
flask. Dilute to volume with Solvent Mixture. Mix!
Filter sufficient volume of the Assay Preparation through a 0.45 m membrane filter into HPLC
vials for injection.
Note: The first few mLs of the filtrate must be discarded before filling the vials. This discarded
volume serves the purpose of saturating the filter.
1
MOBILE PHASE PREPARATION:
Prepare ~1 litre of a mixture of water, methanol and glacial acetic acid (69:28:3). Mix well!
Degas the mobile phase by filtering the mixture through a 0.45 m membrane filter.
Note: Students must measure water and methanol in separate measuring cylinders prior to
mixing in order to obtain accurate volumes. When organic solvents are mixed with aqueous
solvents a change in final volume results: e.g., 690 mL water + 280 mL methanol + 30 mL
glacial acetic acid do not give 1000 mL solvent mixture.
CHROMATOGRAPHIC CONDITIONS:
WAVELENGTH: The liquid chromatograph is equipped with a 275 nm variable wavelength
detector.
COLUMN: C-18 stationary phase maintained at 45 C; Caution: Do not heat (i.e., acquire set-
point) unless mobile phase is flowing through the column.
C-18 column [4.6 mm (id)  10 cm (L), 60 Å, 5 m]
FLOW RATE: 1.0 - 2.0 mL/min (use 1.0 mL/min for ≤3.5 m film thickness)
USP allows flow rate adjustment ± 50%) depending on column back pressure.
Start at 1.0 mL/min.
NB: Check the USP monograph for column dimensions. If your column has reduced length or
inner diameter the USP allows you to use the following equation to recalculate your method flow
rate:
F2 = F1 × l2d22
l1d12
Where
F2, l2, d2 = adjusted flow rate, column length, inner diameter, respectively
F1, l1, d1 = initial flow rate, column length, inner diameter, respectively
INJECTION VOLUME: 10 L
RUN TIME (OR STOP TIME): 8 min (NB: You may adjust stop-time so that all peaks elute within the
chromatographic run-time.)
2
EXPERIMENT #1:
HPLC LABORATORY INJECTION SEQUENCE

STANDARD OPERATING PROCEDURE SOP # PHA533-002
TITLE: PHA 533 HPLC LABORATORY INJECTION SEQUENCE

PURPOSE: This SOP outlines the order for injecting standard and sample preparations into the
HPLC.
SCOPE: This SOP applies to PHA533 Experiment 1
RESPONSIBILITY: Students enrolled in PHA533
RELATED DOCUMENTS: Experiment 1 SUPERCEDES SOP #: New
INJECTION SEQUENCE:
1. Std Blank: Inject once from a standard blank to ensure that the baseline is stable and
no interfering peaks exist on the chromatogram
2. System Suitability + Calibration Stds: Inject 5 times from the 1st standard vial in your
sequence table.
3. Calculate all System Suitability parameters and setup a calibration curve using these
standards (Note: this is done during report writing)
4. Use the average area response value of these standards to quantify your samples
We will not inject a sample blank (Why not? Do we know the excipients used to
manufacture these tablets?)
5. Check Std: Inject once from a separately prepared standard solution.
6. Samples for Exp’t 1 (Assay): Inject once from your sample preparation. (Note to
Instructor: Samples from several students could be injected here.)
7. System Suitability Stds for experiments 1: Inject 5 times from your standard vial.
Note to Instructor: If time does not permit, have students inject once only. In this way they
will be able to calculate all parameters (e.g., Tailing factor, Resolution, etc.), except rsd.
Prepared by: Date:
Checked by: Date:
Approved by: Date:
3
CALCULATIONS:
1) Calculate the Assay Potency for Acetaminophen and Caffeine in tablets or caplets
using the following equation 
% LC (Potency) =
Aspl/Aistd__  ATW  Wstd_balance  DF spl  P
/N Astd/Aistd LC Wspl_balance DF std
P = purity of drug standard = 98.0 %

Aspl = Area of sample
Aistd = Area of internal standard
Astd = Area of standard
/N Astd/Aistd = Average of [standard/internal standard area response] used for system
suitability and calibration
ATW = Average Tablet Weight

Wspl_balance = Weight of sample obtained on the analytical balance
Wstd_balance = Weight of standard obtained on the analytical balance
LC = Label Claim
DF std = Dilution Factor for the standard preparation
DF spl = Dilution Factor for the sample preparation
2) Pass/ fail Acetaminophen and Caffeine assay % LC values (samples 1 and 2) based on the
USP Assay specifications
3) CALCULATE THE UNIFORMITY OF DOSAGE UNITS BY WEIGHT VARIATION:

Calculate the % Label Claim for each or 10 tablets previously weighed using the equation:
% LC (Weight Variation) = % LC for Potency  Individual Weight of each tablet

ATW
4
SUBMISSIONS (students must use a Word processor (e.g., Microsoft word).
1. System Suitability: Create vertical columns and tabulate all system suitability (SS)
results, SS specifications, and pass/fail conclusion for system suitability requirement.
There is no need to show calculations (use a calculator for rsd).
2. Check Standard calculations, results and pass/ fail conclusion. Specifications: % Error
NMT 2.0%.
3. Assay (Expt 1): Tabulate and submit Assay % LC values, assay specification and assay
pass/fail conclusion [You need to show one example of your % LC Assay calculation
and one example of your Weight Variation (% LC) calculations]
4. Weight Variation (WV): Include vertical columns in your table containing (e.g., below)
i) Individual tablet weights
ii) Weight corrected % LC values for 10 units
iii) % LC value, lowest % LC value, highest % LC value, standard deviation (sd) and
rsd for ten % LC values, sd and rsd for 10 tablet weights
iv) Acceptance Value (AV) calculations, USP specifications for uniformity of dosage
unit, conclusion (e.g., pass/fail/etc.)
List all System USP SS % LC by Wt. % LC by Wt. Tablet weights

Suitability (SS) Requirements Variation for Variation for (10 values)
Results including Acetamino- Caffeine
rsd(s) phen (10 values)
(10 values)
Over all Pass/ Fail + conclusion for SS
USP Potency Specification
Sample rsd results

USP Wt. Var. specifications
5
EXPERIMENT #1
TYPICAL CHROMATOGRAM OF ACETAMINOPHEN, CAFFEINE AND BENZOIC ACID
The chromatogram below shows the analyte peaks of interest and the internal standard peak.
Acetaminophen: retention time ~1.3 min
Caffeine: retention time ~1.6 min
Benzoic acid (internal std): retention time ~6.7 min
The peak at ~0.7 min and the negative peak are system peaks arising from the fact that the
mobile phase and the solvent mixture are of different composition.
6
EXPERIMENT #2: % Label Claim by Content Uniformity of
Acetaminophen and Caffeine in tablets
This is a modified USP assay method for Acetaminophen and Caffeine tablets (or
caplets).
Students must consult the latest USP-NF compendium including General Chapters <621> and
<905> for information not contained in this write-up such as specifications and content
uniformity requirements.
[Note: It is wise when conducting HPLC analyses to first prepare the mobile phase and set the
required instrument parameters prior to standard and assay preparations. In this way
instrument stability can be obtained and a stable baseline monitored while wet chemistry is
being done.]

detector.
COLUMN: C-18 stationary phase maintained at 45 C

Caution: Do not heat (i.e., acquire set-point) unless mobile phase is flowing through the
column.
C-18 column [4.6 mm (id)  10 cm (L), 60 Å, 5 m]
FLOW RATE: 1.0 - 2.0 mL/min (use 1.0 mL/min for ≤3.5 m film thickness)
USP allows flow rate adjustment ± 50%) depending on column back pressure.
Start at 2.0 mL/min then change to 1.0 mL/min if the pressure exceeds 310 bar.
inner diameter the USP allows you to use the following equation to recalculate your method flow
rate:
F2 = F1 × l2d22
l1d12
Where
INJECTION VOLUME: 10 L
RUN TIME (OR STOP TIME): 8 min (NB you may adjust stop-time so that all peaks elute within the
chromatographic run-time.)
7
8
Experiment #2: % Label Claim by Content Uniformity of Acetaminophen and Caffeine in tablets

Prepare 1 litre of a mixture of water, methanol and glacial acetic acid (69:28:3).
Mix well!
De-gas the mobile phase by filtering the mixture through a 0.45 m membrane filter.
Note: Students must measure water and methanol in separate measuring cylinders prior to
mixing in order to obtain accurate volumes. When organic solvents are mixed with aqueous
solvents changes in final volume is obtained: e.g., 690 mL water + 280 mL methanol + 30 mL
glacial acetic acid does not give 1000 mL solvent mixture.
INTERNAL STANDARD SOLUTION:

Weigh ~300.0 mg benzoic acid into 50 mL volumetric flask and add methanol to volume.
Sonicate for 1 minute while shaking to dissolve.
SOLVENT MIXTURE:
Prepare sufficient quantity of a mixture of methanol and glacial acetic acid in the ratio 95:5.
Students must go through the entire procedure (prior to entering the lab) and estimate how
much Solvent Mixture will be required.
9
Students: To finish in a timely manner, prepare your samples first. During the 30 min sample
sonication period, turn your attention towards your standard preparations.
STANDARD STOCK SOLUTION:

Weigh 200.0 mg of acetaminophen reference standard and 26.0 mg of caffeine reference
standard into a 100 mL volumetric flask. Add 75 mL of Solvent Mixture and shake (or/and
sonicate) until the standards dissolve. Add Solvent Mixture to volume.
Mix!

Transfer 5.0 mL of the Standard Stock Solution and 3.0 mL of the Internal Standard Solution
into a 50 mL vol. flask and dilute to volume with Solvent Mixture.
Mix!
This solution contains 0.026 mg/mL of caffeine reference standard.
SYSTEM SUITABILITY REQUIREMENTS:

Chromatograph the Working Standard Preparation and record the area responses.
Consult the USP <621> for tailing factor, resolution and rsd equations.
Consult your SOPs for the injection sequence.
[Students are required to perform tailing factor and resolution calculations for peaks on
one standard chromatogram only.]
10
ASSAY (OR SAMPLE) PREPARATION:

Transfer, individually, each of 10 caplets into separate 50 mL volumetric flasks.
If the brand is Tylenol or Excedrin add ~30 - 40 mL of Solvent Mixture to each flask and
sonicate for 30 minutes.
Alternatively, if you have Life brand tablets add ~10 mL of distilled water to each flask and
sonicate for 10 min or until the outer, coloured coating has dissolved. Further add an additional
30 mL (approximately) of Solvent Mixture and continue sonicating for 30 minutes.
Visually inspect each caplet to ensure complete disintegration of the white tablet core
into white sediment. If not then continue to sonicate until caplet disintegration has been
achieved.
Dilute the contents in each volumetric flask with Solvent Mixture to volume. Mix!
Transfer 1.0 mL of this solution and 3.0 mL of the Internal Standard Solution to 50 mL vol.
flasks. Dilute to volume with Solvent Mixture. Mix.
Filter through a 0.45 m membrane into HPLC vials.

Caffeine concentration = 0.026 mg/mL.
11

HPLC.
RELATED DOCUMENTS: Experiment 2 SUPERCEDES SOP #: New
INJECTION SEQUENCE:
1. Std Blank: Inject once from a standard blank to ensure that the baseline is stable and
no interfering peaks exist on the chromatogram
2. System Suitability + Calibration Stds: Inject 5 times from the 1st standard vial in your
sequence table.
3. Calculate all System Suitability parameters and setup a calibration curve using these
standards (Note: this is done during report writing)
4. Use the average area response value of these standards to quantify your samples
5. Check Std: Inject once from a separately prepared standard solution.
6. Samples for Exp’t 2: Inject once from each of your 10 sample preparations.
7. System Suitability Stds for experiments 1: Inject one std (time does not allow us to
inject 5 times from your standard vial).
Prepared by: Date:
Checked by: Date:
Approved by: Date:
12
CALCULATIONS:
i. Calculate the Uniformity of acetaminophen and Caffeine in each Dosage Units by
Content Uniformity; this is done by calculating the % Label Claim for
acetaminophen and cafeine in each caplet.
Equation:
% LC (Content Uniformity) =
Aspl/Aistd  Wstd_balance  DFspl  P
/N Astd/Aistd LC DFstd
P = purity of drug standard = 98.0%

Aspl = Area of sample
Aistd = Area of internal standard
Astd = Area of standard
/N Astd/Aistd = Average of [standard area response /internal standard area response]
used for system suitability and calibration
Wstd_balance = Weight of standard obtained on the analytical balance
LC = Label Claim
DF std = Dilution Factor for the standard preparation
DF spl = Dilution Factor for the sample preparation
ii. Calculate the USP Acceptance Value.

iii. Based on the USP specifications pass and fail your results.
13
SUBMISSION REQUIREMENTS: Experiments 1 & 2 require a joint discussion
by each student (provided at the end of experiment 2).
The information given below is provided as a guide only, and is based on results that have been
submitted in previous years!
Ultimately, each student will have different sets of data and this could lead to individualized
discussion.
Discuss your results in a coherent manner.
Based on the USP requirements for Uniformity of Dosage Form, discuss whether your tablets
should be analysed by a) Weight Variation or b) Content Uniformity.
Do look for trends as you browse data from Weight Variation to Content Uniformity. Observe
whether an increase or decrease in a particular set of data is common for both Acetaminophen
and Caffeine. In this way you will be able to ascertain that the trend is real rather than random.
Discuss why that particular trend is observed and provide a logical scientific explanation for your
observation or otherwise, explain why your data goes against the expected trend.
Examples of trends to look for (once again, this is based on past submissions…your data may
show different or/and additional trends) as you go from WV to CU:
 Average %LC
 Gap between highest and lowest %LC values which is also indicated by your
sample rsd
 Comparison of system rsd vs WV sample rsd vs CU sample rsd values vs tablet
weight rsd
14
SUBMISSION REQUIREMENTS
5. No need to calculate and submit System Suitability requirements such as RRT, R, Tf,
etc. Assume that the system is suitable for the purpose of this discussion. However, do
list the rsd for your 5 replicate standard injections (i.e., your system rsd).
6. Include a table (use a word processor e.g., Microsoft word) listing and comparing results
obtained (see below) from both Weight Variation and Content Uniformity tests (you do
not have to show calculations)
E.g.,
Weight Variation Content Uniformity

System rsd (from 5 stds): System rsd:
% LC % LC
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
Average % LC Average % LC
Lowest % LC Lowest % LC
Highest % LC Highest % LC
sd for ten % LC values sd (% LC)
rsd (% LC) rsd (% LC)
sd for 10 tablet weights
rsd for 10 tablet weights
AV AV
L1 L1
Conclusion about the Conclusion about the
uniformity of dosage units uniformity of dosage units
Discussion:
 Discussion must be at least ¾ page and typed, using a narrative style!
 Students must demonstrate that they can gather facts using references beyond
the course manual/notes and USPNF.
 Please list these references.
 Please do not use your discussion to re-write the procedure.
 Please do not use your discussion to re-results without explanation.
 Please do not discuss the impact of petty and avoidable analyst errors (e.g.,
sample spillage, wrong volume measurements or incorrect mass weighed).
 Do not use your discussion section to blame your partner (everyone within a
group is responsible for the group’s results).
15
 Everyone must submit individual reports!
TYPICAL CHROMATOGRAM OF ACETAMINOPHEN, CAFFEINE AND BENZOIC ACID
The chromatogram below shows the analyte peaks of interest and the internal standard peak.
Acetaminophen: retention time 0.652 min
Caffeine: retention time 1.000 min
Benzoic acid (internal std): retention time 3.158 min
The peak at 0.493 min and the negative peak are system peaks arising from the fact that the
mobile phase and the solvent mixture are of different composition.
16
EXPERIMENT #3: Determination of Percent Salicylic Acid impurity in Aspirin Tablets (325
mg)


HPLC.
RELATED DOCUMENTS: Experiments 3 SUPERCEDES SOP #: New
INJECTION SEQUENCE:
1. Std Blank: Inject once from a standard blank to ensure that the baseline is stable
and no interfering peaks exist in the chromatogram
2. System Suitability + Calibration Stds: Inject the number of System Suitability
standards required by the USP
3. Calculate all System Suitability parameters and setup a calibration curve using
these standards
4. Use the average area response value of these standards to quantify your
samples
5. Check Std: Inject once from a separately prepared standard solution
6. Inject once from your sample blank (SB) preparation (if SB preparation is
required by your instructor)
7. Inject once from your sample preparation
8. Inject the number of System Suitability standards required by the USP
Prepared by: Date:
Checked by: Date:
Approved by: Date:
17
[Note: It is wise when conducting HPLC analyses to first prepare the mobile phase and set the
required instrument parameters prior to standard and assay preparations. In this way
instrument stability can be obtained and a stable baseline monitored while wet chemistry is
being done.]
detector.
COLUMN: C-18 (stationary phase, 1.5 – 10 µm particle size), 4.0 mm (id)  30 cm (L)
FLOW RATE: 2.0 mL/min
inner diameter then use the following equation to recalculate your method flow rate:
F2 = F1 × l2d22
l1d12
Where
INJECTION VOLUME: 10 L NB: Reduce your injection volume if there are signs of column
overload (e.g., peak fronting!!); injection volume can be reduced to a point that allows us to
maintain adequate precision and detection limits (ref. USPNF)
RUN TIME: 10 min NB: you may reduce stop-time so that all peaks elute within the
chromatographic run-time.

Dissolve 2 g of sodium 1-heptanesulfonate in a mixture of 850 mL of water. Add 150 mL of
acetonitrile and mix. Adjust pH to 3.4 with glacial acetic acid while stirring.
Note:
i) The pH of the aqueous mix only (before addition of acetonitrile) is ~2.8. Addition of organic
solvents raise pH value (compared to pH of aqueous only mixtures. Students should do a
literature review of this concept.
ii) Students must measure water and acetonitrile in separate measuring cylinders prior to mixing
in order to obtain accurate volumes. When organic solvents are mixed with aqueous solvents
changes in final volume is obtained: e.g., 720 mL aqueous solvent + 280 mL organic solvent
does not give 1000 mL solvent mixture.
DILUTING SOLUTION:
Prepare a 500 mL of a mixture of acetonitrile and formic acid in the ratio 99:1.
18
mg)
STANDARD PREPARATION
Dissolve 100 mg of aspirin reference standard in Diluting solution in a 200 mL volumetric flask.

Dissolve 15.0 mg of Salicylic Acid reference standard in Standard Preparation in a 100 mL
volumetric flask.
Mix!
Transfer 5.0 mL to a 50 mL vol. flask. Dilute to volume with Standard Preparation.
Mix!
This solution contains 0.015 mg/mL of Salicylic Acid reference standard.
PRELAB QUESTION:
Why is it necessary to prepare a Working Standard Preparation containing aspirin drug
substance even though we are only analyzing the salicylic acid content in the caplet?
SYSTEM SUITABILITY REQUIREMENTS:

Chromatograph the Working Standard Preparation and record the area responses.
The tailing factor for each analyte peak is not more than (NMT) 1.2.
The resolution, R, between salicylic acid and aspirin is not less than (NLT) 2.0.
Consult the USP <621> for tailing factor, resolution and rsd equations.
Consult your SOPs for the injection sequence.
[Students are required to perform tailing factor and resolution calculations for peaks on
one chromatogram only.]
The relative standard deviation (rsd) of the salicylic acid peak area responses for replicate
injections is NMT 4.0%.
PRELAB QUESTION:
How many injections "replicate" refers to? Answer: Consult the USP General Chapters <621>
for System Suitability requirements.
19
mg)
SAMPLE BLANK OR PLACEBO: (INSTRUCTOR WILL INFORM YOU WHETHER TO PREPARE THIS, OR NOT)
ASPIRIN TABLET FORMULATION

Ingredients (purity) Amounts (mg)
Aspirin d.s. (98.0%) 331.63
Microcrystalline cellulose 100
Total tablet weight 431.63
Prepare a sample blank (i.e., containing placebo) based on i) formulation above, ii) sample
preparation procedure below, iii) your understanding of the USP (Notices) definition of a ‘Blank
Determination’
20
mg)
TEST (OR SAMPLE ) PREPARATION:

Weigh altogether 20 tablets and calculate the average tablet weight (ATW).
Crush 5 tablets (please return the additional 15 tablets to the original container) and mix the
tablet powder using a mortar and pestle.
Transfer an amount of tablet powder equivalent to 100 mg aspirin into a 100 mL vol. flask. Add
20.0 mL of Diluting solution, and shake mechanically for 10 minutes. Mix!
This solution contains 5 mg/mL of Aspirin.
Filter sufficient volume of the Assay Preparation through a 0.45 m membrane filter into the
HPLC vial for injection.
PRELAB QUESTIONS:
1. The USP gives the relative retention times (rrt) of 0.7 for salicylic acid and 1.0 for
aspirin. What is the expected rt for salicylic acid if aspirin elutes at 12 minutes?
2. Calculate the Limit of SA as a percent wt/wt value.
21
mg)
CALCULATIONS:
Calculate the % FSA impurity in aspirin tablet using the equation
% Impurity =
Aimpspl  Wimpstd_balance  DFimpspl  ATW  P
Aimpstd Wtabletspl_balance DFimpstd LC

N
Where P is the purity of the FSA Standard = 98.0
Aimpspl = Area of FSA in sample ,
Aimpstd = Area of FSA in standard,
 = Average standard area response used for system suitability

N
Wtabletspl_balance = Weight of tablet powder sample obtained on the analytical balance
Wimpstd_balance = Weight of impurity reference standard
LC = label Claim (325 mg)

ATW = Average tablet weight
DF = dilution factor
22
EXPERIMENT #3
Determination of Percent Salicylic Acid impurity in Aspirin Tablets (325 mg)
SUBMISSION REQUIREMENTS
1) Calculate
i. % FSA
ii. % of each unknown impurity (show calculations for one impurity, only)
iii. Total % unknown impurities in Aspirin tablets.
2) Please show the basic equation used to calculate % unknown impurity even if
you have no unknown impurity in your sample.
Ensure that symbols for standards are unambiguous. E.g., use Wt FSA_std and Wt ASA_std to
denote the weight of FSA and weight of ASA in your standard preparation, respectively.
Note: All unknown impurities are expressed as a % of the active pharmaceutical ingredient.
Therefore, % unknown impurity may be derived from the following expression:
% Unknown Impurity =
Conc. of unknown imp. obtained from the experiment ×100
Concentration of active in the sample preparation
E.g., Using the symbol Cunk_imp_exp’t for conc. of unknown imp. obtained from the experiment, and
CASA_STD for the conc. of aspirin in the standard preparation.
Then, Cunk_imp_exp’t α Areaunk_imp_sample
CASA_std α AreaASA_std
Therefore, Cunk_imp_exp’t = Areaunk_imp_sample × CASA_std

AreaASA_std
Next, express CASA_std and Concentration of active in the sample preparation in terms of Wt and
DF (dilution factor) to obtain your final equation.
Note the following:
 Any peak present in your sample chromatogram (except the drug) but not in your
sample blank chromatogram is an impurity peak
 Any peak in your sample chromatogram with a higher area count than that found in
your sample blank chromatogram (similar retention times to 1 decimal place)
represents impurities in your sample. You must subtract the area counts between
sample and sample blank, then calculate the % impurities
23
Presenting your data:
E.g., Table 1: Impurities in Aspirin Tablet
Retention time % Impurity

for each imp. Report to one decimal
(min) place greater than that in
(Sample 1) your specification
3.45 (FSA)
4.50 (unk.)
5.28 (unk.)
Total unknown
Imputities
Quote all specifications and pass/ fail the batch.
3) Answer the following questions:

i. Show how sodium 1-heptanesulfonate (anionic or negatively charged pic)
interacts with salicylic acid to form ion-pair compounds. Draw structures, and all
three equilibria as part of your answer.
ii. Review the literature and name 1 cationic (i.e., positively charged pic) pic
reagent. Show how it interacts with an analyte to form an ion pair.
iii. Review the literature and list 2 factors that may affect the retention times of an
analyte during ion paired chromatography.
24
mg)
TYPICAL CHROMATOGRAM OF SALICYLIC ACID AND ASPIRIN

Note that the column used to acquire this chromatogram is no longer being used, therefore
retention times will be much smaller with the shorter columns now being used. However, the
order of elution for FSA and ASA will remain the same.
Many of the earlier eluting peaks originate from solvent and reagent impurities and may not be
seen on your chromatogram, depending on the purity of solvent and reagents used.
 Free Salicylic Acid (FSA) also called Salicylic Acid (SA): Retention time 6.98 min
 Aspirin (also called Acetyl Salicylic Acid or ASA): Retention time 10.03 min
25
EXPERIMENT #4: DETERMINATION OF RESIDUAL SOLVENT IMPURITIES (RSIS)
IN ASPIRIN TABLETS (USP <467> HEADSPACE GC METHOD)
Procedure A (PEAK IDENTIFICATION) CHROMATOGRAPHIC CONDITIONS GC SET-UP:
Column: HP-624, 30 m (Long) ´ 0.32 mm (ID), film thickness 1.8 mm conditioned from 40-280
°C until baseline stabilizes
Carrier Gas: helium, linear velocity = 35 cm/sec, constant flow mode
Sample Inlet Port configuration:

Split mode
Injection port temperature 140 °C

Split Ratio: 0.2:1 (i.e., 1:5)
Septum purge: 3.0 – 5.8 (3.0 for older GCs, 5.8 for newest GC [variable septum purge) closer to
the door]
Detector gases configuration:
Flame Ionization Detector (FID) temperature 250 °C

FID Hydrogen flow rate: 40 mL/min
FID Air flow rate: 400 mL/min (450 mL/min for newest GC closer to door)
FID Constant Column + Make-up Gas (Helium): 40 mL/min
GC Oven Temperature settings:

Temperature Programming
Initial Temp: 40 °C
Hold Time: 20 min
Rate or ramp: 10 °C/min
2nd Temp: 240 °C

Hold Time: 20 min
Post Run Temp: 40 °C

Hold Time: 3 min
26
EXPERIMENT #4: Determination of Residual Solvent Impurities (RSIs) in Aspirin tablets
(USP <467> Headspace GC Method)
Procedure A: HEADSPACE CONDITIONS:

{Note: It is wise when conducting GC analyses to first set all required GC instrument
parameters [e.g., gas (carrier, make-up and detector) flows, temperature, etc.] prior to standard
and sample preparations. In this way instrument stability can be obtained through conditioning
of the column and a stable baseline monitored while wet chemistry is being done.}
Instructor (for the HSGC closest to the door):
 Carrier gas flow is controlled by the pressure gauge on the wall (Manual
Pressure Control, MPC) and by a valve on the Headspace instrument. Verify that
Helium is flowing out of the end of transfer line, forming bubbles in a beaker of
water
 Vial pressure is controlled by the EPC (Aux 3), located on the GC instrument.
Turn on the Aux He pressure and set to 15 Psi
ZONE TEMP:
Vial/ Oven: 80 °C
Loop: 90 °C
Transfer Line: 105 °C
EVENT TIMES:
GC Cycle Time: 70 min

Vial Equilibrium Times: 45 min (i.e., time required to heat and equilibrate sample vial)
Vial Pressurization Time: 1.0 min (i.e., time required to pressurize vial with helium to 10-15 psi)
Loop Fill Time: 1.00 min
Loop Equilibrium Time: 1.0 min
Injection Time: 1.0 min
(Note: Only 1 Injections per vial)
VIAL PARAMETERS: (FOR NEWER HSGCS) USE CHEMSTATION TO SET UP SEQUENCE
27
Procedure A: STANDARD AND SAMPLE PREPARATION
PREAMBLE:
During HPLC analysis we oftentimes prepare the standard solutions and establish system
suitability before preparing our sample or test preparations. This is done so that sample,
reagents and solvents are not wasted in the event that system suitability fails. Also, this is an
effective approach towards analysis when dealing with unstable samples such as antibiotics.
Obviously, for such samples (antibiotics) we would wish to do our sample preparation just prior
to injection to ensure that analyte degradation in the diluting solvents does not occur while the
“run” is in progress.
With Headspace-GC analysis, the solvents undergoing analysis are mostly stable so there is no
need to prepare our samples just prior to a GC chromatographic run.
Due to solvent volatility, however, it is quite easy to lose method accuracy and precision if the
standards and samples are not prepared in the same manner. This requires that both
standards and samples be prepared at the same approximate time in order to minimize
variations arising from the fluctuations in ambient temperature during the course of the working
day.
General Instructions:
 Prepare standards and samples for Procedure A and Procedure B
simultaneously! Both procedures require the same vials, therefore simply prepare
duplicates of each vial); crimp to seal all vials immediately!
 Your instructor will set up online GC methods!
 Students in the same lab section (e.g., section A) will create/run a single
sequence using the GC equipped with DB 624 column (Procedure A) [Instructor:
calculate overall runtime to determine if instruments is ready for your next lab
session (~1 hr per vial]
 Students from another lab section (e.g., section B) will create/ run one big
sequence using the GC equipped with DB Wax column (Procedure B)
[Instructor: calculate overall runtime to determine if instruments is ready for your
next lab session (~1 hr per vial]

28
EXPERIMENT #4
Determination of Residual Solvent Impurities (RSIs) in Aspirin tablets (USP <467>
Headspace GC Method)
Procedure A—Please read the next section, ”INJECTION SEQUENCE, SYSTEM SUITABILITY
REQUIREMENTS” to know how many vials of each standard you should prepare.
Omit all Class 1 preparations
1) Class 1 Standard Stock Solution— (Prepared by Technician)

Prep. i: Transfer 1.0 mL of USP Class 1 Residual Solvents Mixture RS into a 100-
mL volumetric flask previously filled with 9 mL of dimethyl sulfoxide. Dilute with
water to volume, stopper and mix.
Prep. ii: Using an Eppendorf pipette transfer 1.0 mL of Prep. i into a 100-mL
volumetric flask previously filled with approximately 90 mL of distilled water. Dilute
with water to volume, stopper and mix.
Prep. iii: Using an Eppendorf pipette transfer 1.0 mL of Prep. ii to a 10-mL

volumetric flask containing ~8 mL distilled water. Dilute with water to volume,
stopper and mix.
This final preparation (Prep. iii) is your Class 1 Standard Stock Solution. (Post-lab
Question: Calculate the concentration in µg/mL).
1) Class 1 Standard Solution— Transfer exactly 5.0 mL of distilled water into the
headspace vial. Using an Eppendorf pipette deliver 1.0 mL of Class 1 Standard
Stock Solution into the headspace vial. Apply the cap. Crimp to seal tight. Mix.
(Post-lab Question: Calculate the amount of residual solvent impurities in
µg).
2) Class 2 A Standard Stock Solution — (Prepared by Technician)

Using an Eppendorf pipette deliver 1.0 mL of USP Residual Solvents Class 2—Mixture
A RS to a 100-mL volumetric flask previously filled with approximately 90 mL of distilled
water. Dilute with water to volume, stopper and mix. This is your Class 2 Standard Stock
Solution A. (Post-lab Question: Calculate the concentration in µg/mL).
29
EXPERIMENT #4
Class 2 B Standard Stock Solution— (Prepared by Technician)
Using an Eppendorf pipette deliver 1.0 mL of USP Residual Solvents Class 2—Mixture B
RS to a 100-mL volumetric flask previously filled with approximately 90 mL distilled
Solution B. (Post-lab Question: Calculate the concentration in µg/mL).
3) Class 2 Mixture A Working Standard Solution— Transfer exactly 5.0 mL of

distilled water into the headspace vial. Using an Eppendorf pipette deliver 1.0 mL
of Class 2 Standard Stock Solution A to the headspace vial. Apply the cap. Crimp
to seal tight then mix. (Post-lab Question: Calculate the amount of residual
solvent impurities in µg).
4) Class 2 Mixture B Working Standard Solution— Transfer 5.0 mL of Class 2

Standard Stock Solution B into the headspace vial. Using an Eppendorf pipette
add 1.0 mL of water. Apply the cap. Crimp to seal tight and mix. (Post-lab
Question: Calculate the amount of residual solvent impurities in µg).
5) Test Solution— Weigh 0.050 g of sample into the headspace vial, and add 6.0
mL of water. Apply the cap. Crimp to seal tight then sonicate for 2 min.
30
EXPERIMENT #4
6) Class 1 System Suitability Solution— Weigh 0.050 g of sample into the

headspace vial, and transfer exactly 5.0 mL of distilled water. Using an
Eppendorf pipette deliver 1.0 mL of Class 1 Standard Stock Solution into the
headspace vial. Apply the cap. Crimp to seal tight then sonicate for 2 min.
 Unlike HPLC, we must inject only once from each headspace vial. Why?
 Note: Gently apply a twisting pressure to the metallic cap on the headspace vial
using your thumb and forefinger to be sure the seal is good. Caps should not
turn!
 Calculate the Reporting Limit (RL) for each residual solvent impurity.
Show one example of your RL calculations, only.
A useful equation for calculating RL (except for 1,1,1-trichloroethane: Please see

“Criteria for Reporting Limits” in the next section to understand RL for 1, 1, 1-
trichloroethane)
RL (ppm, wt/wt or µg/g, except for 1,1,1-trichloroethane) =
Amount of residual solvent impurity in the standard vial (µg)

Amount of sample in the test vial (g)
E.g., of how to present your results
Name of Class Reporting (Average) area Area in Is the RSi in your

2A RSi Limit in Standard Sample sample below the
Reporting Limit
Name of Class Reporting (Average) area Area in Is the RSi in your

2B RSi Limit in Standard Sample sample below the
Reporting Limit
31
Procedure A: GC-HEADSPACE INJECTION SEQUENCE, SYSTEM SUITABILITY

REQUIREMENTS, AND REPORTING LIMIT
 Inject one standard blank (to ensure that the baseline is stable and no interfering
peaks exist on the chromatogram)
 Inject three Class 1 Standard Solution {need three vials!}; for i) system suitability
and ii) for reporting limits {use average peak areas}
RSD ≤ 7.0
Signal-to-noise ratio (S/N) of 1,1,1-trichloroethane in the Class 1 Standard Solution

is ≥ 5
 Inject one Class 1 System Suitability (SS) Solution for system suitability only;
Signal-to-noise ratio of each peak is ≥ 3
 Inject one Class 2 Mixture A Standard Solution for i) SS and ii) for reporting limits
Resolution, R, between acetonitrile and methylene chloride is ≥ 1.0
 Inject one Class 2 Mixture B Standard Solution for reporting limits, only
 Inject one test solution {for i) peak identification based on retention times and ii)
to evaluate whether the residual solvents exceed the reporting limits. We will not
inject a sample blank (Why?)}
Criteria for Reporting Limits: Except 1,1,1-trichloroethane, the peak area responses
for all other solvents in Classes 1, 2A and 2B standards correlate to residual solvent
concentrations at the reporting limit.
Criteria for 1,1,1-trichloroethane Reporting Limits: 150 × peak area response for
1,1,1-trichloroethane in Class 1 standard correlate to the concentration of 1,1,1-
trichloroethane at the reporting limit. Therefore,
1) if a peak response of any peak (except 1,1,1-trichloroethane) in the test

solution is ≥ to a corresponding peak in Class 1 standard, or Class 2A or
2B mixture standards, then proceed to Procedure B for confirmation
or
32
2) if a peak response of 1,1,1-trichloroethane in the test solution is ≥ (150 ×
peak corresponding to 1,1,1-trichloroethane in Class 1 standard), then
proceed to Procedure B for confirmation
3) if a peak response of any peak (except 1,1,1-trichloroethane) in the test

solution is less than a corresponding peak in Class 1 standard, the drug
product passes the requirements for residual solvents.
4) For 1,1,1-trichloroethane, peak response in the test solution must be less

than 150 × peak area corresponding to 1,1,1-trichloroethane in Class 1
standard, then the drug product passes that requirement for residual
solvents.
 Due to the exorbitant cost of USP standards, we will not be running any system
suitability at the end of the sequence.
PRELAB QUESTION:
STUDENTS: WRITE AND SUBMIT YOUR HEADSPACE-GC INJECTION SEQUENCE TO YOUR INSTRUCTOR
(IF REQUESTED).
HEADSPACE-GC INJECTION SEQUENCE FORMAT FOR SUBMISSION
VIAL # NAME OF RSI NUMBER OF INJECTIONS

2 CLASS 2A ×1
33
Procedure B: CHROMATOGRAPHIC CONDITIONS GC SET-UP:
Column: HP-Wax, 30 m (Long) ´ 0.32 mm (ID), film thickness 0.25 mm conditioned from 40-280
°C until baseline stabilizes
Carrier Gas: helium, linear velocity = 35 cm/sec, constant flow mode
Sample Inlet Port configuration:

Split mode
Injection port temperature 140 °C

Split Ratio: 0.2:1 (i.e., 1:5)
Septum purge: 3.0 or 5.8 (3.0 for older GCs, 5.8 for newest GC [variable septum purge] closer
to the door)
Detector gases configuration:
Flame Ionization Detector (FID) temperature 250 °C

FID Hydrogen flow rate: 40 mL/min
FID Air flow rate: 400 or 450 mL/min (450 for newest GC closer to the door)
FID Constant Column + Make-up Gas (Helium): 40 mL/min
GC Oven Temperature settings:

Temperature Programming
Initial Temp: 50 °C
Hold Time: 20 min
Rate or ramp: 6 °C/min
2nd Temp: 165 °C

Hold Time: 20 min
Post Run Temp: 50 °C

Hold Time: 3 min
34
Procedure B: HEADSPACE CONDITIONS

{Note: It is wise when conducting GC analyses to first set all required GC instrument
parameters [e.g., gas (carrier, make-up and detector) flows, temperature, etc.] prior to standard
and sample preparations. In this way instrument stability can be obtained through conditioning
of the column and a stable baseline monitored while wet chemistry is being done.}
ZONE TEMP:
Vial/ Oven: 80 °C
Loop: 90 °C
Transfer Line: 105 °C
EVENT TIMES:
GC Cycle Time: 65 min

Vial Equilibrium Times: 45 min (i.e., time required to heat and equilibrate sample vial)
Vial Pressurization Time: 1.0 min (i.e., time required to pressurize vial with helium to 10-15 psi)
Loop Fill Time: 1.00 min
Loop Equilibrium Time: 1.0 min
Injection Time: 1.0 min
(Note: Only 1 Injections per vial)
VIAL PARAMETERS 1: (FOR NEWER HSGCS) USE CHEMSTATION TO SET UP SEQUENCE
VIAL PARAMETERS 2: (FOR OLDEST HSGC) ENTER CONDITIONS USING THE HEADSPACE CONSOLE
BUTTONS
First Vial: 1
Last Vial: enter the vial # of your last vial
Shake: High (3)
Shake High (min): 3
35
EXPERIMENT #4
Procedure B: STANDARD AND SAMPLE PREPARATION

(PREPARE ALL STANDARDS AND SAMPLES AT THE SAME TIME)
During HPLC analysis we oftentimes prepare the standard solutions and establish system
suitability before preparing our sample or test preparations. This is done so that sample,
reagents and solvents are not wasted in the event that system suitability fails. This is also an
effective approach when dealing with unstable samples. For such samples, we wish to prepare
our sample just prior to injection to ensure that analyte degradation in the diluting solvents does
not occur while the “run” is in progress.
With Headspace-GC analysis, the residual solvents being analysed are stable, so there is no
need to prepare our samples just prior to a GC chromatographic run. Due to solvent volatility, it
is quite easy to decrease method accuracy and precision if standards and samples are not
prepared at the same approximate time. This minimizes variations arising from the fluctuations
in ambient temperature during the course of the working day.
Procedure B— Please read the next section (”INJECTION SEQUENCE, SYSTEM SUITABILITY
REQUIREMENTS”) to know how many vials of each standard you should prepare.
Omit all Class 1 preparations
1) Class 1 Standard Stock Solution— (Prepared by Technician)

Prep. i: Transfer 1.0 mL of USP Class 1 Residual Solvents Mixture RS into a 100-mL
volumetric flask previously filled with 9 mL of dimethyl sulfoxide. Dilute with water to
volume, stopper and mix.
Prep. ii: Using an Eppendorf pipette transfer 1.0 mL of Prep. i into a 100-mL volumetric
flask previously filled with approximately 90 mL of distilled water. Dilute with water to
volume, stopper and mix.
Prep. iii: Using an Eppendorf pipette transfer 1.0 mL of Prep. ii to a 10-mL volumetric
flask containing ~8 mL distilled water. Dilute with water to volume, stopper and mix.
This final preparation (Prep. iii) is your Class 1 Standard Stock Solution. (Post-lab
Question: Calculate the concentration in µg/mL).
36
EXPERIMENT #4
2) Class 1 Standard Solution— Transfer exactly 5.0 mL of distilled water into the
headspace vial. Using an Eppendorf pipette deliver 1.0 mL of Class 1 Standard
Stock Solution into the headspace vial. Apply the cap. Crimp to seal tight. Mix.
(Post-lab Question: Calculate the amount of residual solvent impurities in
µg).
3) Class 2 A Standard Stock Solution — (Prepared by Technician)

Using an Eppendorf pipette deliver 1.0 mL of USP Residual Solvents Class 2—Mixture
A RS to a 100-mL volumetric flask previously filled with approximately 90 mL of distilled
Solution A. (Post-lab Question: Calculate the concentration in µg/mL).
Class 2 B Standard Stock Solution— (Prepared by Technician)
Using an Eppendorf pipette deliver 1.0 mL of USP Residual Solvents Class 2—Mixture B
RS to a 100-mL volumetric flask previously filled with approximately 90 mL distilled
Solution B. (Post-lab Question: Calculate the concentration in µg/mL).
4) Class 2 Mixture A Working Standard Solution— Transfer exactly 5.0 mL of

distilled water into the headspace vial. Using an Eppendorf pipette deliver 1.0 mL
of Class 2 Standard Stock Solution A to the headspace vial. Apply the cap. Crimp
to seal tight then mix. (Post-lab Question: Calculate the amount of residual
solvent impurities in µg).
37
EXPERIMENT #4
5) Class 2 Mixture B Working Standard Solution— Transfer 5.0 mL of Class 2

Standard Stock Solution B into the headspace vial. Using an Eppendorf pipette
add 1.0 mL of water. Apply the cap. Crimp to seal tight and mix. (Post-lab
Question: Calculate the amount of residual solvent impurities in µg).
6) Test Solution— Weigh 0.050 g of sample into the headspace vial, and add 6.0
mL of water. Apply the cap. Crimp to seal tight then sonicate for 2 min.
7) Class 1 System Suitability Solution— Class 1 System Suitability Solution—

Weigh 0.050 g of sample into the headspace vial, and transfer exactly 5.0 mL of
distilled water. Using an Eppendorf pipette deliver 1.0 mL of Class 1 Standard
Stock Solution into the headspace vial. Apply the cap. Crimp to seal tight then
sonicate for 2 min.
 Unlike HPLC, we must inject only once from each headspace vial. Why?
 Note: Gently apply a twisting pressure to the metallic cap on the headspace vial
using your thumb and forefinger to be sure the seal is good. Caps should not
turn!
38
Procedure B: GC-HEADSPACE INJECTION SEQUENCE, SYSTEM SUITABILITY

REQUIREMENTS, REPORTING LIMIT OR THRESHOLD
 Inject one standard blank (to ensure that the baseline is stable and no interfering
peaks exist on the chromatogram)
 Inject three Class 1 Standard Solution {need three vials!}; for i) system suitability
and ii) for reporting limits {use average peak areas}
RSD ≤ 7.0
Signal-to-noise ratio (S/N) of benzene in the Class 1 Standard Solution is ≥ 5
 Inject one Class 1 System Suitability (SS) Solution for system suitability only;
Signal-to-noise ratio of each peak is ≥ 3
 Inject one Class 2 Mixture A Standard Solution for i) SS and ii) for reporting
limits
Resolution, R, between acetonitrile and cis-dichloroethene is ≥ 1.0
 Inject one Class 2 Mixture B Standard Solution for reporting limits
 Inject one test solution {for i) peak identification based on retention times and ii)
to evaluate whether the residual solvents exceed the reporting limits. We will not
inject a sample blank (Why?)}
Criteria for Reporting Limits: Except 1,1,1-trichloroethane, the peak area responses
for all other solvents in Classes 1, 2A and 2B standards correlate to residual solvent
concentrations at the reporting limit.
Therefore, if any identified residual solvent (RS) peak area (in the Test vials from
Procedure A) ≥ corresponding RS peak area in Class 1, Class 2A or 2B mixture
standards (Procedure B), then proceed to Procedure C for quantification,
otherwise the drug product passes the requirements for residual solvents.
Criteria for 1,1,1-trichloroethane Reporting Limits: 150 × peak area response for
1,1,1-trichloroethane in Class 1 standard correlate to the concentration of 1,1,1-
trichloroethane at the reporting limit.
39
EXPERIMENT #4
IMPORTANT: REFER TO APPENDIX 12 FOR CHROMATOGRAMS OF ALL RSI STANDARDS
TYPICAL CHROMATOGRAM of Procedure A - Class 2A RSI standard chromatogram
40
EXPERIMENT #4
Lab 4 submission requirements
1) Print the following chromatograms:

Procedure A
i. Class 2A + standard blank chromatogram + test chromatogram; all with the same
response scale and time scale
ii. Class 2B + standard blank chromatogram + test chromatogram; all with the same
Procedure B
i. Class 2A + standard blank chromatogram + test chromatogram; all with the same
ii. Class 2B + standard blank chromatogram + test chromatogram; all with the same
2) Number and identify peaks corresponding to residual solvent impurities in the

following chromatograms:
Procedure A
i. Class 2A + standard blank chromatogram + test chromatogram
ii. Class 2B + standard blank chromatogram + test chromatogram
Procedure B
i. Class 2A + standard blank chromatogram + test chromatogram
ii. Class 2B + standard blank chromatogram + test chromatogram
41
EXPERIMENT #4
NB: Requirements: Annotate RSi peaks on your chromatograms using numbers then write (on
an area below your chromatographic peaks) RSi names corresponding to each numbered peak.
You will need to use the following chromatograms to assist with the identification of RSi peaks
(if any) on your Test chromatogram:
a) Standard chromatograms
b) Standard blank chromatogram
3) Calculate the Reporting Limits (ppm or µg RSi per g tested material) for benzene,
acetonitrile and any RSi found in your sample.
Note: Vial preparations for Class 2A is different from 2B.
REPORTING LIMIT (ppm) =

Amount of RSi in your STD vial required by the method (µg)
Amount of sample in your Test vial required by the method (g)
USP Class 1, 2A, and 2B reference standard mixtures, used to prepare stock solutions
(Experiment 4), are prepared by dissolving residual solvent impurities (RSI) in dimethyl
sulfoxide. Concentrations are given on the next page.
Report your RL values in the format shown below. Give one example of your RL calculations
only, using benzene.
USP RSi reference standard Reporting Limits, g RSi

per g sample
benzene
acetonitrile
Any RSi found in your sample
42
RESIDUAL SOLVENTS USED TO MAKE STOCK STANDARDS
Ampoules containing residual solvents impurities (RSis) were purchased (i.e., USP standards)
and used by the technician to prepare Class 1, Class 2A and Class 2B stock standards. The
concentration of each RSi (dissolved in 1 mL dimethyl sulfoxide) contained within ampoules are
given below:
Residual Solvents, Class 1

Table 1: Concentration of each RSi dissolved in 1 mL dimethyl sulfoxide (1 mL/ampul)
Benzene 10 mg/mL
Carbon tetrachloride 20 mg/mL
1,2-Dichloroethane 25 mg/mL
1,1-Dichloroethene 40 mg/mL
1,1,1-Trichloroethane 50 mg/mL
Residual Solvents, Class 2 - Mix A

Acetonitrile 2.05 mg/mL
Chlorobenzene 1.80 mg/mL
Cyclohexane 19.40 mg/mL
cis-1,2-Dichloroethene 4.70 mg/mL
trans-1,2-Dichloroethene 4.70 mg/mL
1,4-Dioxane 1.90 mg/mL
Ethylbenzene 1.84 mg/mL
Methanol 15.00 mg/mL
Methylcyclohexane 5.90 mg/mL
Methylene chloride (dichloromethane) 3.00 mg/mL
Tetrahydrofuran 3.45 mg/mL
Toluene 4.45 mg/mL
m-Xylene 6.51 mg/mL
o-Xylene 0.98 mg/mL
p-Xylene 1.52 mg/mL
Residual Solvents, Class 2 - Mix B

n-Hexane 1,450 μg/mL
Chloroform 300 μg/mL
1,2-Dimethoxyethane 500 μg/mL
2-Hexanone 250 μg/mL
Nitromethane 250 μg/mL
43
Pyridine 1,000 μg/mL
Tetralin 500 μg/mL
Trichloroethene 400 μg/mL
Lab 4 submission
1) Identify (by retention time) all residual solvent impurities (RSis) in your test
chromatogram by comparing retention times of your sample against standard and
blank.
2) Conclude if any RSi in your sample exceeds the reporting level (RL). Recall that
standards are prepared at the RL and that the area of any RSi in the standard
represents the reporting level. If the area for any RSi in your sample exceeds that in
your standard then you must proceed to Procedure B for confirmation.
Note the following:

• Please prepare a table containing information from class 2A chromatograms [i)
STDs, ii) Test and iii) Blank] using the format shown (Table 1, next page).
• Refer to Appendix 13 in this manual for chromatograms showing RSis in USP
Class 2A standard solution.
• Due to small run-to-run variations in temperature and flow rate, your retention times
may be fractionally faster or slower than those in your manual. Do your best to
identify RSis in your chromatograms, despite these real-life variations. You will have
to make an individual judgment call.
• Note: Large amounts of RSi in your test chromatogram may result in peak shape
distortion (i.e., distortion from Gaussian shape) which could shift peak apex and
therefore cause slight retention times shifts.
• HSGC systems may vary in sensitivity. As a result, some peaks may be barely
discernable (e.g., 1,4-dioxane) compared to what is in your manual.
• Area in Test chromatogram must be corrected for Blank contributions: E.g., if your
blank and sample have areas 10 and 12 units at similar retention times, then it
means that your sample really has only 12 – 10 = 2 area counts. I.e., 10 of 12 units
came from your solvents used in both sample and blank and so should be
discounted.
44
Table 1 (provided as an example only):
Procedure A— Class 2A standards chromatogram (do same for Procedure B—
Class 2A standards)
All RSis in Class 2A Peaks in Blank Peaks in Test: Conclusion

STD prep. (RT, min), (RT, min), Area corrected for
Areas (RL) Areas area in Blk
(RT, min), Areas
e.g., Methanol (2.306 (2.7), 130 (2.307 min), 25830 A) Identified as MeOH
min), 11.2 – 130 = 25700 B) Area exceeds that in
standard, therefore proceed
to Procedure B
Acetonitrile Below reporting level, do not
proceed to Pro. B
Conclusion: (E.g., Batch passes/ batch fails, etc.)
45
EXPERIMENT #4
E.G., OF REPORTING LIMIT (RL) CALCULATIONS:
REPORTING LIMIT =
Amount of RSi in your STD vial required by the method (µg)
Amount of sample in your Test vial required by the method (g)
Step 1: Calculate the amount (µg) of RSi in your standard vial

Step 2: Know the amount crushed Aspirin tablets sample (g) in your test vial
Step 3: Express the amount of RSi as ppm (Rsi expressed as a concentration of your crushed
Aspirin tablet sample, Wt per Wt ) to obtain the Reporting Limit
Example of Calculations (Note that USP residual solvents, e.g., benzene is sold in glass
ampoules, dissolved in dimethyl sulfoxide)
The following concentrations are given:
RSi Solvents dissolved in DMSO Conc (ppm, Weight / Volume)

Benzene 10,500
Carbon tetrachloride 20,000
1,2-Dichloromethane 25,000
1,1- Dichloroethene 40,000
1,1,1-Trichloroethane 50,000
 Since we are given: USP Carbon tetrachloride has a concentration (in DMSO) of
10,000 ppm (i.e., 10,000 µg per mL)
 Then supposing 1.0 mL USP Carbon tetrachloride was transferred to a 100 mL

volumetric flask containing water, this flask would contain 10,000 µg carbon
tetrachloride or 100 µg per mL
 If we transferred 1.0 mL of this solution (from the volumetric flask) to a

headspace vial containing 5.0 mL water, then the amount of carbon tetrachloride
in the vial = 100 µg (This is Step 1)
46
 Step 2: Know the amount of crushed Aspirin tablet sample in your test vial. In this
example I will use 0.1 g as the amount for crushed tablet sample (Students: You
must use the amount from actual experiment)
 Step 3: Express the amount of RSi as a ppm (wt per wt) concentration of the
sample
47
E.g.
Reporting Limit = 100 µg (carbon tetrachloride in standard vial)
0.1g (i.e., amount of crushed Aspirin tablets)
= 1000 µg in 1 g sample = 1000 ppm (Wt / Wt)
Conclusion:
Reporting Limit (RL) for carbon tetrachloride = 1000 ppm
Please note that RL values are expressed as ppm!
Further explanation:
 If 0.1 g of your crushed tablet sample contains 1000 ppm of carbon

tetrachloride it will generate the same area count as the standard.
 Therefore the amount of carbon tetrachloride in your sample is at the

reporting limit.
 Samples with carbon tetrachloride area counts above that of the standard
area count have amounts of carbon tetrachloride exceeding the Reporting
Limit. The USP requires that these samples proceed to quantitation via
Procedure C and the sample results must be reported (regardless of whether
they are above or below the Reporting Limit. This is true for all residual
solvents. Any residual solvent in your sample that exceeds the RL must be
quantitated via Procedure C and be reported.
 Samples with area counts below that of the standard area counts have
amounts below the Reporting Limit for carbon tetrachloride. No quantitation
via Procedure C is required. This is true for all residual solvents. Any residual
solvent in your sample that is below the RL need not be quantitated via
Procedure C or be reported.
48

2021 Labs 1,2,3,4 - Students

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EXPERIMENT #1:

INTERNAL STANDARD SOLUTION:

STANDARD STOCK SOLUTION:

WORKING STANDARD PREPARATION:

ASSAY (I.E., SAMPLE) PREPARATION:

C-18 column [4.6 mm (id)  10 cm (L), 60 Å, 5 m]

HPLC LABORATORY INJECTION SEQUENCE

TITLE: PHA 533 HPLC LABORATORY INJECTION SEQUENCE

Prepared by: Date:

Checked by: Date:

Approved by: Date:

Aspl/Aistd__  ATW  Wstd_balance  DF spl  P

/N Astd/Aistd LC Wspl_balance DF std

P = purity of drug standard = 98.0 %

ATW = Average Tablet Weight

3) CALCULATE THE UNIFORMITY OF DOSAGE UNITS BY WEIGHT VARIATION:

% LC (Weight Variation) = % LC for Potency  Individual Weight of each tablet

List all System USP SS % LC by Wt. % LC by Wt. Tablet weights

Over all Pass/ Fail + conclusion for SS

USP Potency Specification

Sample rsd results

WAVELENGTH: The liquid chromatograph is equipped with a 275 nm variable wavelength

COLUMN: C-18 stationary phase maintained at 45 C

MOBILE PHASE PREPARATION:

INTERNAL STANDARD SOLUTION:

STANDARD STOCK SOLUTION:

WORKING STANDARD PREPARATION:

SYSTEM SUITABILITY REQUIREMENTS:

ASSAY (OR SAMPLE) PREPARATION:

Filter through a 0.45 m membrane into HPLC vials.

TITLE: PHA 533 HPLC LABORATORY INJECTION SEQUENCE

5. Check Std: Inject once from a separately prepared standard solution.

Checked by: Date:

Approved by: Date:

Aspl/Aistd  Wstd_balance  DFspl  P

/N Astd/Aistd LC DFstd

P = purity of drug standard = 98.0%

ii. Calculate the USP Acceptance Value.

Weight Variation Content Uniformity

TYPICAL CHROMATOGRAM OF ACETAMINOPHEN, CAFFEINE AND BENZOIC ACID

HPLC LABORATORY INJECTION SEQUENCE

TITLE: PHA 533 HPLC LABORATORY INJECTION SEQUENCE

Checked by: Date:

Approved by: Date:

MOBILE PHASE PREPARATION:

WORKING STANDARD PREPARATION:

SYSTEM SUITABILITY REQUIREMENTS:

ASPIRIN TABLET FORMULATION

TEST (OR SAMPLE ) PREPARATION:

Aimpspl  Wimpstd_balance  DFimpspl  ATW  P

Aimpstd Wtabletspl_balance DFimpstd LC

 = Average standard area response used for system suitability

LC = label Claim (325 mg)

Therefore, Cunk_imp_exp’t = Areaunk_imp_sample × CASA_std

Retention time % Impurity

Quote all specifications and pass/ fail the batch.

3) Answer the following questions:

TYPICAL CHROMATOGRAM OF SALICYLIC ACID AND ASPIRIN

Procedure A (PEAK IDENTIFICATION) CHROMATOGRAPHIC CONDITIONS GC SET-UP:

Sample Inlet Port configuration:

Injection port temperature 140 °C

Detector gases configuration: