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Gravimetric Sample PreparationReducing Sample Size and OOS
Errors
Contents
1 What is gravimetric sample preparation?
2 Source and impact of OOS results
3 GWP The Weighing Standard
3.1 Science Based Compliance
3.2 Measurement Uncertainty and Minimum Weight
3.3 Safety Factor to Compensate for Variability
4 Potential errors in sample preparation
4.1 Types of laboratory variability
4.2 Use of volumetric glassware
4.3 Mixing
4.4 Labelling
5 Gravimetric sample preparation
5.1 Gravimetric sample preparation equipment
5.2 Effect on diluent addition
5.3 Effect on process security (data management)
5.4 Effect on process efficiency
5.5 How can a gravimetric approach reduce the sample volume?
5.6 Volumetric vs. gravimetric comparison
6 Conclusion
7 References
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aper 1 What is gravimetric sample preparation?
The definition of gravimetric sample preparation is weighing not
only the solid but also the solvent to enable a specific
concentration to be prepared accurately and precisely.
Gravimetric sample preparation offers a revolutionary new
alternative way to prepare samples and standards which dramatically
reduces the variability in the samples and has a positive impact on
the instances of out-of-specification results.
2 Source and impact of Out-of-Specification Results
Out of Specification (OOS) results have had a significant impact
in the pharmaceutical industry for many years, but in particular
since a court ruling in 1993. In this case, the court ruled in
favor of Barr Labs which upheld their view that an OOS result does
not necessarily constitute a batch failure (1). They felt that an
OOS result should be investigated to determine if there are other
causes such as a laboratory error. However, the court did not like
the way Barr Labs was conducting their laboratory investigations.
As a consequence, the FDA updated their guide-lines concerning how
to handle an OOS result and how to perform a proper investigation
in October 2006 (2). Since then, the FDA has issued a significant
number of 483 observations concerning poor investigations.
Furthermore, in the FDA guidance concerning OOS investigations, the
FDA states that:
Laboratory errors should be relatively rare. Frequent errors
suggest a problem that might be due to inadequate training of
analysts, poorly maintained or improperly calibrated equipment, or
careless work.
However, as there are a significant number of FDA 483
observations relating to poor investigations, the incidence of
laboratory errors may not be as rare as it should. Unfortunately,
there is no published data which shows that for every OOS result
generated there were many more minor errors that didnt lead to an
OOS result. These errors may have been classified as a Note to
Record, or simply noted in the laboratory notebook as an error.
Many companies dont investigate these errors even though they are
probably symptoms of potentially more serious issues with the
analysis method or process.
An article was published about 10 years ago in LC/GC magazine
concerning OOS results (3). The article discussed two aspects of
laboratory work: firstly, what are the sources of OOS errors and
secondly, what activi-ties consume laboratory personnels time in an
analytical workflow (Full Time Equivalent (FTE) spend in the
laboratory).
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Time Spent in Lab Sources of OOS Errors
Data Management (27%) Sample Introduction (6%)
Chromatography (7%)
Integration (6%)
Instrument (8%)
Calibration (9%)
Contamination (4%)
Columns (11%)
Operator (19%)
Sample Processing (61%)Sample Processing (30%)
Collection (6%)
Analysis (6%)
Figure 1: Sources of OOS results and time spent in
laboratory
The results from this survey indicated that the two largest
sources of OOS results are sample processing followed by human
error and the most time-consuming (manual) task is sample
processing (Figure 1). Even though no follow-up survey has been
published since, it appears that these results are still valid
today. In fact, the instru-mentation, data systems, and columns
have improved significantly during the last 10 years, whilst the
sample processing has remained essentially the same. Since these
other developments have saved a significant amount of time in the
workflow, it is fair to assume that the sample processing aspect of
the laboratory work is likely to account for even more than 61% of
the FTE. Figure 2 illustrates the accepted formal process for how
to investigate OOS results:
Figure 2: Formal process for an OOS investigation.
Confirm Variation in Manufacturing
Process
Variation or Error in Manu-facturing or Sampling
Retesting According to SOP or Protocol
Repeat Test to Substitute the OOS Result
No further OOS resultsAdditional OOS Result occur
Retesting Error in Sampling Procedure and Resample
Lab Error Identified ?
Laboratory InvestigationChecklist Approach: Investigation of
standards used,
analytical techniques, etc.
Out-of-Specification Result
Stop Production Release
YesNo
No
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aper A great deal of effort is expended when a sample generates
an OOS result and there have been numerous arti-cles published on
how this process should work. Obvious OOS results can take three
days of work but serious
ones can take months of work to resolve. The cost can easily run
into many thousands or tens of thousands of dollars. Given the
large impact that an OOS result has on a pharmaceutical company,
the best course of action should be to put every effort into
avoiding them in the first place.
Besides trying to determine the root cause, the other
significant issue seems to be the mounting Corrective and
Preventative Actions or CAPAs that a company generates over a
number of years as a result of these laboratory investigations.
These CAPAs typically cause procedural changes to SOPs and other
documents and over time they become unmanageable and difficult to
follow which has the potential to cause even more issues.
The overriding problem with CAPAs is that, in the vast majority
of cases, the assumption tends to be made that it is an isolated
incident and so only a specific item in a workflow or process is
addressed. In other cases, there is a tendency to blame a single
employee or a simple laboratory error. In some cases, this simple
error may be the only thing that that needs to be addressed but in
many, if not most cases, it is the whole process or workflow that
needs to addressed instead of one step. This is especially true of
sample preparation in the laboratory.
3 GWP The Weighing Standard
3.1 Science Based Compliance
Before we elaborate further on the sample preparation process
and its individual steps, we would like to highlight the scientific
background of one of the most important steps of the whole process
weighing. Weighing is a key activity in most laboratories but it
isnt always sufficiently understood and its complexity is often
underestimated. As the quality of weighing strongly influences the
quality of the whole sample and standard preparation process, USP
specifically requires in its General Chapter highly accurate
weighing results used for quantitative analysis (4).
Repeatability is satisfactory, if two times the standard
deviation of the weighed value, divided by the nominal value of the
weight used, does not exceed 0.10%"
Such a stringent requirement is not implemented for other
instruments, where quite often the analytical develop-ment group
sets the method requirements.
State-of-the-art strategies for adhering to consistently
accurate and reliable weighing processes comprise of sci-entific
methodologies on balance selection and testing (5, 6, 7). The
weighing standard GWP takes these prin-ciples into account, and
furthermore addresses typical misconceptions on weighing which are
very widespread in the industry. One of them is that many users
believe what you see is what you get. What do we mean by that? Let
us make an example: A user weighs a standard on a XP205 semi-micro
balance and gets a reading of 50.13 mg which he believes is the
true amount of material that he was weighing. However, this reading
might not exactly reflect the amount weighed, in other words, the
amount weighed might differ slightly from the indication. This is
due to the so-called measurement uncertainty which is inherent for
every instrument you might think of.
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3.2 Measurement Uncertainty and Minimum Weight
Measurement uncertainty of instruments is determined in
calibration, and the results issued in appropriate cali-bration
certificates. In general, measurement uncertainty of weighing
systems can be approximated by a straight line the higher the load
on the balance, the larger the (absolute) measurement uncertainty
becomes, as shown in Figure 3.
Figure 3: Measurement Uncertainty: Absolute (green line) and
relative (blue line) measurement uncertainty of a weighing
instrument. The accuracy limit of the balance, the so-called
minimum weight, is the intersection point between relative
measurement uncertainty and the required weighing accuracy.
Looking at the relative measurement uncertainty, which is the
absolute measurement uncertainty divided by the load, and usually
indicated in per cent, we clearly see that the smaller the load is,
the larger the relative measurement uncertainty becomes. If you
weigh at the very low end of the balances measurement range, the
relative uncertainty can become so high that the weighing result
cannot be trusted anymore. It is good practice to define accuracy
(tolerance) requirements for every weighing process. For
quantitative analysis this is even stipulated by USP General
Chapter . Weighing in the red area as indicated in the figure will
result in inaccu-rate measurements, as here the measurement
uncertainty of the instrument is larger than the required accuracy
of the weighing process. Consequently, there is a specific accuracy
limit for every weighing instrument the so-called minimum sample
weight, or short, minimum weight, and you have to weigh at least
this amount of sample in order to have a sufficiently small
uncertainty that satisfies the specific weighing accuracy
requirement.
While measurement uncertainty is described in much detail in the
respective literature (8, 9), we want to emphasize that for
weighing small loads on analytical and microbalances and samples
and standards usu-ally are small loads as compared to the capacity
of the balance the dominant contribution factor to weighing
uncertainty stems from repeatability (expressed as the standard
deviation of a series of weighings). This is also reflected in USP
General Chapter as discussed above.
Even though adherence to this USP requirement seems to be
straightforward, many companies still have issues with the correct
interpretation. While environmental influences and operator
variability which contribute to inde-terminate errors and
consequently to possible changes or fluctuations of the reading of
a weighing device are discussed later, another misconception which
is prevalent in the industry is briefly discussed now. Many
compa-nies wrongly believe that the weight of the tare also
accounts for the adherence to the minimum weight. In other words,
if the tare weighs more than the minimum weight, any sample
quantity can be added and USP is automatically fulfilled. This
would mean that with a large enough tare container you might
believe that you could even weigh a microgram on a 5-place balance
and still comply with the uncertainty requirement of 0.1%. Such an
extreme example clearly shows us that this widespread
misinterpretation indeed does not make any sense. For this reason
USP has attempted to clarify this issue in the latest revision of
General Chapter (10):
The minimum weight applies to the sample weight, not to the tare
or gross weight.
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aper 3.3 Safety Factor to Compensate for Variability
The minimum weight of balances is furthermore not constant, but
varies over time. This is due to changing environmental conditions
that affect the performance of the instrument, as for example
vibrations or draft. The operator himself also adds variability to
the minimum weight, as different people might weigh differently or
with a different skill level on the balance. In order to ensure
that you always weigh above the minimum weight as determined at
calibration (at a particular time with particular environmental
conditions by a particular qualified person), it is highly
recommended to apply a safety factor, see Figure 4.
Figure 4: Safety Factor: Variability of the relative measurement
uncertainty due to changing environmental conditions and influences
intro-duced by the operator. Weighing in the green area guarantees
adherence to the weighing accuracy requirements (application of a
safety factor).
The safety factor describes that you would only weigh
sufficiently above the minimum weight as determined at calibration.
For manual weighing, a safety factor of 2 is commonly used,
provided you have reasonably stable environmental conditions and
trained operators. For very critical applications or a very
unstable environment, an even higher safety factor is
recommended.
Later in this paper we will elaborate more in detail about
typical minimum weight values and recommended safety factors for
automated gravimetric sample preparation as compared to manual
weighing (see 5.5 How can a gravimetric approach reduce the sample
volume?).
4 Potential errors in sample preparation
4.1 Types of Laboratory Variability (Errors)
To be able to deal with variability in the laboratory, it is
important to first understand the types of variability and where
they occur. Variability in the data generated comes from two
sources, determinate and indeterminate errors. A determinate error
has a definite direction and magnitude and has an assignable cause,
their cause can be determined. Determinate error is also called
systematic error. Determinate errors can (theoretically) be
elimi-nated through instrument adjustments. Indeterminate errors
are also called random errors, or noise. Indetermi-nate errors can
be minimized but cannot be eliminated. Examples of these types of
errors are described in more detail in 5.6 Volumetric vs.
gravimetric comparison.
The largest cause of indeterminate errors in the laboratory is
from manual operations where the human element is involved. Figures
1 highlights the issue that sample processing and human operations
are the biggest source of laboratory errors.
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For a typical simple sample preparation workflow, Figure 5
demonstrates the number of steps involved.
Material and Equipment
Gathering materials volumetric glassware, standards, reagents
and samples Check balance cleanliness, minimum weight requirements,
calibration date Check other instruments for
calibration/qualification dates and record Prepare diluents
combining solvents in correct proportions, pH adjusting, etc.
Record manufacture, lot number, expiration dates for all materials
Labeling hand written or generated
Sonicating and QSing
Fill with specific amount of diluent, mix/sonicate and cool to
room temperature QS with diluent Successive dilution, if required,
QS and mix Record data and label volumetric flasks Transfer to
vials and label vials Repeat steps for each preparation
Weighing and Labeling
Tare weigh container Perform weighing operation by: carefully
adding material into weighing container closing door and allowing
balance to settle repeat until target weighing obtained Record
weight and carefully transfer powder to volumetric Reweigh
container, perform net weight calculation, dispose of container
Label all in a compliant manner, safety and GMP
Analyze and Clean Up
Place vials in instrument and perform analysis Volumetric flasks
(after results are verified): rinse with solvent followed by water
remove any labelling with solvent Have volumetric flasks and
pipettes transported and washed Retrieve clean volumetric flasks
and pipettes, place in storage Re-order ones that didnt make it
back from dishwasher or were damaged
Figure 5: Simple sample preparation workflow
The steps are grouped into four distinct activities. The first
concerns gathering materials and ensuring the equip-ment is clean
and calibrated. There are a number of steps at the beginning that
would not necessarily cause an OOS result but would certainly
feature in both GxP and safety audits. Resolving these audit issues
can consume a significant amount of time and effort and should also
be avoided as they may lead to future OOS results.
The second activity involves weighing and labeling steps.
Typically, these are manual time consuming opera-tions and the
weighing steps can contribute to OOS results. An additional
difficulty is that these steps are manu-ally intensive, so it can
potentially be very difficult to determine the root cause of this
operation.
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aper Following weighing, the diluent must be added. This is
followed by dissolution and any successive dilutions that may be
required. The weighing, sonication and QSing steps are repeated for
each standard and sample. Dilution
steps are a big potential source of errors, and consequently
this activity will be discussed in more detail in the section
below. Finally, the samples are analysed and the materials and
equipment are tidied up. This requires disposal of unused
solutions, rinsing of flasks and pipettes, and other resupply
steps.
Therefore, a simple sample preparation takes ten or more steps
with an additional ten miscellaneous steps. If two standards and a
sample were prepared, approximately 40 steps would need to be
carried out. A 40 step process has a significant number of areas
where problems could potentially occur. Furthermore, some of these
steps can be expanded and a detailed analysis with more complex
steps, including operations such as extrac-tion and filtering,
might result in a process with one hundred or more steps. Given
this number of manual steps where indeterminate errors occur, it is
surprising that OOS results are not even more frequent.
Fortunately, many but not all of these errors are found before the
final results are obtained but they do significantly impact the
pro-ductivity of the laboratory operation and the overall quality
of the data.
4.2 Use of volumetric glassware for diluent addition
Some of the key steps in sample preparations involve the use of
volumetric glassware. The production process which created flasks
with accuracies similar to those in use today has been in operation
for the last 75 years. It is astonishing to consider that sample
preparation methods have not advanced signifi-cantly during the
last century. Especially when compared to the advances in
instrumentation and software that have provided dramatic
improvements in analysis and data processing.
What are some of the errors that are associated with volumetric
glassware?
4.2.1 Failure ratesA paper published by Coleman and Harris from
NIST in 2005 (11) states the failure rates of new glassware to meet
Class A specifications have been found to be as high as 50%. This
finding may not be too surprising since there are a variety of
glassware suppliers, some of them having very cut rate pricing.
This simply illustrates the importance of proper evaluation of
glassware before selecting a vendor, and the conclusion that a
supplier should not be chosen on price alone.
4.2.2 Calibration temperatureA volumetric flask is typically
calibrated for use at 20C. Any temperature change of the solution
which results in a volume change can cause errors if the working
temperature is significantly away from the volumetric calibra-tion
point of 20C. These temperature deviations may be caused by
endothermic or exothermic mixing of solvents. In addition, a
sonicator which is often used to aid dissolution of solids, can
cause a significant increase in the temperature of the solutions.
If care is not taken to return the flask and contents back to 20C,
errors can be introduced. Additional information regarding the
operating temperature ranges for Class A glassware can be found in
a UKAS publication on traceability of volumetric apparatus
(12).
4.2.3 Contamination risksA significant problem with reusing any
item in the laboratory such as volumetric glassware is
contamination from other products or reagents. It is very
difficult, if not impossible, to qualify a glassware washing system
in development due to the number and variability of the products
tested. Therefore it is very important to always pre-rinse and post
rinse the glassware with the appropriate solvents to minimize this.
Unfortunately, this leads to solvent waste and is time
consuming.
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There have been a number of OOS investigations where the analyst
has forgotten to pre-rinse his volumetric flasks. In fact, this
implies that another analyst must have forgotten to perform a post
rinsing operation too. The problem created by this repeating issue
is how to justify a CAPA that says retraining is addressing the
problem when in fact is doesnt. How do you know which analyst didnt
do the post-rinsing, do you retrain all analysts? If people
continue to forget, what are the next steps? Do you spend hundreds
of thousands of dollars on a system to try and remove the
contaminants from the glassware? Some companies have. But does it
really make sense to spend that much money on 100 year old
technology?
4.2.4 RevalidationColeman and Harris also suggested in their
paper (11) that the calibration of the glassware should be verified
at least every 10 years. This could potentially be a very expensive
process considering that the number of volumet-ric flasks in a
typical pharmaceutical analytical or QA/QC department can be very
large. It could potentially be cheaper to throw them all away and
start again.
4.2.5 TolerancesIn Table 1, the published NIST relative percent
errors associated with each size of volumetric glassware are
listed. In each case as the size (volume) of the glassware
decreases, the error risk (tolerance) increases significantly.
Pipettes FlasksVolume (mL) Relative % Error Volume (mL) Relative
% Error
1 0.60 5 0.40
2 0.30 10 0.20
3 0.33 25 0.12
4 0.25 50 0.10
5 0.20 100 0.08
10 0.20 200 0.05
Table 1: Relative percent errors for Class A glassware
Any errors associated with the Class A glassware that does not
meet the specification would be even larger, as mentioned
previously in 4.2.1 concerning the high failure rates.
Aside from the significant increase in the relative percent
error, the smaller glassware is also very technique dependent when
it comes to manual manipulations. For example, in one company, it
was found that many of the analysts could not properly use a
pipette below 2 mL and errors as high as 5% were noted in some
cases (13).
4.2.6 CostThe cost of using volumetric glassware is an issue
that is often taken for granted. A great deal of effort goes into
keeping glassware organized and stored in a laboratory. Everyone
who has worked in the lab has probably been charged with ordering
and putting away the clean glassware at some point in their career.
This is costing a proportion of a FTE. The pre and post rinsing on
a company wide basis, assuming a very conservative 25 mL use per
flask and 10,000 sample preparations, might be costing the company
$10,000 or more each year at a $40/liter average solvent cost.
These annually recurring costs can add up to a substantial amount,
especially when you include the rising cost of waste disposal.
Additional costs can be incurred by lab services groups that
transport the flasks to and from the washing facility; and
attrition due to breakage and damage, which results in approx. 10%
loss each year at a cost of about $20 per flask.
4.3 Mixing Most samples are sonicated to expedite the breakup of
tablets, capsules, or powders. Sonication can cause OOS results
when there is a lack of robustness in the method. The lack of
robustness arises from the improper use of the sonicator and
whether or not the instrument is tuned properly. Most sonicators
have the following instruc-tions on them:
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Donotplacepartsorcontainersdirectlyonthebottomofthecleaningtank:useatryorwiretosuspenditems.
Donotallowthesolutiontodropmorethat3/8inchbelowtheoperatinglevellinewiththecleaneron.
However, from industry feedback it seems that few people follow
those instructions. The pictures in Figure 6 illustrate the
difference between a tuned sonicator on the left and an untuned
sonicator on the right.
Figure 6: Foils from a tuned and untuned sonicator
The untuned system has most of its energy focused in the middle
of the bath, where you can see the large hole in the foil (on the
right). Therefore, the energy of the system can vary significantly
depending on the placement of the sample into the bath.
Many methods need to have better instructions for the final
mixing step. Most methods only state to mix well without realizing
that a volumetric flask is an extremely poor mixing vessel that
requires it to be inverted a num-ber of times to ensure proper
mixing.
4.4 Labeling
Labeling can potentially cause OOS results due to label mix ups
but the most significant issues with labeling are usually
identified at safety and GxP audit times. Regardless of what a
labeling SOP in the company states, when flasks in laboratories are
examined, the labeling content usually ranges from the absolute
minimum to the very detailed. Of course, all of these permanent
marker labels must be removed before sending them out to be washed
and that necessitates the use of methanol or acetone to wipe down
the flasks which takes time and wastes more solvent.
5 Gravimetric sample preparation
It is universally accepted that a gravimetric measurement is
intrinsically more accurate that a volumetric mea-surement. In
fact, pipettes and volumetric measuring equipment are calibrated
using gravimetric methods. So why are people still weighing solids
and powders on a weighing paper, transferring them into volumetric
flasks, and subjectively reading the meniscus to prepare a specific
concentration?
Gravimetric sample preparation, which involves automated
weighing and dispensing of the solid and of the diluent, can reduce
laboratory errors and increase laboratory efficiency (14).Using
this method, weighing papers, weighing boats and volumetric flasks
are no longer necessary.
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5.1 Gravimetric sample preparation equipment
There are a number of systems available which support the
technique of gravimetric sample preparation. These are described in
more detail in this section and listed in Table 2. In all of the
systems, a gravimetric approach is used to deliver both powders and
liquids into a small, disposable target container (vial) which is
positioned on a semi-micro or micro balance (see Figure 7).
Figure 7: Gravimetric dispensing systems: QB5 on the left; QX1
on the right
The user has to define the concentration required and the target
amount of solution to be prepared. The software calculates the
target amount of solid to dispense and then, according to the
actual amount of solid dispensed, delivers the appropriate amount
of diluent to prepare an extremely accurate and precise
concentration. Individual dosing heads are used for each substance.
The powder dosing heads are disposable and designed for use with a
single substance only. The solvent dispensing heads are also
exclusively used for a single solvent, and no valves or switching
or washing of lines is employed. This means that all potential
sources of cross contamina-tion risk are eliminated.
The QB3 and QB5 systems automate the powder and liquid
dispensing process. The difference between the QB3 and QB5 system
is the type of balance integrated, and hence the weighing
specifications. These differences are described in Table 2. For
both the QB3 and QB5 systems, the appropriate powder dispensing
head has to be selected and manually inserted into the system. Each
dosing head is identified by RFID chip for process security. This
head has to be manually removed and replaced by the appropriate
liquid dosing head at the Add diluent step. All data is recorded
electronically and can be automatically printed onto labels.
For fully automated sample preparation approach the QX7 is
available. The QX7 can accommodate 10 individ-ual powder dosing
heads and 5 different solvent bottles on the system at any one
time. Dispensing of solid and liquid can occur unattended, with the
system automatically selecting and replacing the appropriate heads
and placing target vials onto the balance position for each
programmed sample preparation. Again heads are tracked by RFID
chip, target vials are identified by barcode and the data is stored
electronically.
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aper QB3 QB5 QX7Minimum weight 10 mg 6 mg 1.4 mg
Readability 0.01 mg 0.005 mg 0.001 mg [1 g]
Number of sample preparations in a batch
1 1 30 1) 1 48 (4 racks of 12)
Minimum concentration 0.01 mg/g (GxP) 0.01 mg/g (GxP) 0.01 mg/g
(GxP)
Maximum concentration 1 g/g 1 g/g 1 g/g
Max amount that can be prepared 100 g 100 g ~10 g
Number of solids online 1 1 10
Number of solvents online 1 1 5
1) If the 30-position autosampler is integrated
Table 2: Specifications of Quantos QB3, QB5 and QX7 Gravimetric
Dispensing Systems
These automated gravimetric systems are being adopted by
analytical and QA/QC laboratories in the pharma-ceutical
industry.
5.2 Effect on diluent addition
We have already discussed how the addition of the diluent by
volumetric dosing introduces a manifold of inde-terminate handling
errors, such as reading the meniscus incorrectly or using the
glassware at temperatures where thermal expansion causes the limit
of error to be exceeded. Gravimetric liquid dosing avoids these
non-quantifiable handling errors, and furthermore weighing liquids
at gram levels is very accurate because it results in a completely
negligible measurement uncertainty contribution of this process
step. The amount of diluent is typically far above the minimum
weight of the balance, where the hyperbolic shape of the relative
measurement uncertainty curve flattens out to almost zero (see
Figure 3).
With gravimetric sample preparation the exact amount of
substance dispensed (whether dispensed manually by spatula or using
an automated dosing head) is recorded and used to accurately
calculate the amount of solvent to weigh in to the container. Any
under or overshoot in powder weighing doesnt require you to waste
time add-ing a tiny amount more or scooping material off the
weighing paper with your spatula. The automated liquid dispensing
compensates for this and delivers the correct amount of diluent to
achieve the required concentration. The sample can then be
sonicated and used without the need to be concerned about
temperature and mixing.
5.3 Effect on process security (data management)
^In terms of data management there are also distinct advantages
of automated gravimetric sample preparation in comparison with the
manual volumetric approach. The manual approach requires hand
transcription which has a high error-risk, and it relies on the
diligence of each individual analyst. It is simply not possible to
digitally record which size of volumetric flask was used
automatically.With an automated approach the data transcription is
automated. All samples and solvents are identified by RFID (radio
frequency identification) to eliminate the possibility of weighing
the wrong sample. All weighed samples are documented electronically
(target weights, actual weights and concentrations achieved) and
the data is fully traceable.Labels with pre-defined fields can be
printed automatically for immediate application to the vial
containing the prepared solution. This addresses the issues with
accuracy and consistency of labeling which were discussed in the
context of the manual approach.
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5.4 Effect on process efficiency
The simplified gravimetric sample preparation workflow is shown
in Figure 8. This workflow has approximately 30% fewer steps than
the volumetric workflow, described in Figure 3, which is a
significant reduction. This makes the process much more efficient
and results in a significant amount of time saved in the sample
prepara-tion workflow. More importantly, the steps that have
greatest potential to cause OOS results have been elimi-nated.
Material and Equipment
Gathering materials standards, reagents and samples Check
balance cleanliness, minimum weight requirements, calibration date
Check other instruments for calibration/qualification dates and
record Prepare diluents combining solvents in correct proportions,
pH adjusting, etc. Labeling handled by system using RFID
technology
Sonicating and QSing
Sonicate, no QSing required Successive dilution, if required,
using solvent dosing head Information and labels handled
automatically Transfer to vials and label vials Repeat steps for
each preparation
Weighing and Labeling
Place powder dosing head on instrument, deliver target amount
Place solvent dosing head on instrument, deliver target amount
Information and labels automatically recorded
Analyze and Clean Up
Place vials in instrument and perform analysis Dispose of
containers
Figure 8: New simplified sample prep workflow using a
gravimetric approach
5.5 How can a gravimetric approach reduce the sample volume?
Let us now look at the typical USP minimum weight and the
recommended safety factor for Quantos automated gravimetric dosing
systems as compared to manual weighing systems. Provided the same
weighing module is used in both instruments, generally the minimum
weight for the Quantos system is significantly lower as com-pared
to the equivalent conventional weighing system. One main reason is
that environmental effects espe-cially drafts and temperature
differences between balance and sample are more efficiently
prevented when using Quantos. Furthermore, the variability
introduced by the operator is completely removed. The exclusion of
the operator variability and the efficient compensation of
environmental effects allows for applying a smaller
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14 White Paper METTLER TOLEDO
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typically 1.5 instead of 2 within a controlled labora-tory
environment with trained operators. Consequently, sample sizes can
be chosen much smaller for Quantos,
typically smaller by a factor of 3 as compared to manual
weighing. While usually 50 mg are weighed manually on a XP205
semi-micro balance, a Quantos QB5 using the same technology
typically allows for weighing only 13.5 mg.Besides of the smaller
minimum weight and the smaller safety factor, an additional benefit
of gravimetric sample preparation is that the analyst is not
constrained to make a volume based on the size of volumetric flask
avail-able. These two factors combined mean that smaller amounts of
sample can be used, smaller volumes of solu-tions can be prepared,
less solvent is consumed and there is less waste to dispose of. The
automated nature of the process also makes it safer for the
analyst.
Figure 9: Comparison of volumetric and gravimetric sample
preparation on the amount of solvent used.
Being constrained to Class A volumetric glassware forces an
analyst to use much more substance than neces-sary because they are
limited to the capacity of the flasks available. The amount of
solvent used when preparing samples using a manual volumetric
approach is indicated by the red line in Figure 8. The sharp
vertical drops in the red line on the graph indicate the four
discrete points at which the minimum weight of the substance
matches an available volumetric flask size (10ml, 25ml, 50ml,
100ml) and thus the only concentrations at which the actual minimum
weight can be used to achieve the desired concentration. In all
other cases, the amount of sample must be rounded up to match the
next size of volumetric flask available.
Automated gravimetric sample preparation is not limited to these
discrete intervals, as the smooth green curve indicates. With this
method, the minimum amount of sample (9 mg) can be weighed at every
point on the curve, and the corresponding amount of solvent can be
added, to achieve the required concentration. At the point
indicated by the blue arrow (which corresponds to a concentration
of 1.1 mg/ml), six times less substance and solvent is used to
achieve the same concentration gravimetrically. This is due to an
equal contri-bution from both of the factors mentioned previously:
the minimum amount of sample that can be weighed on the Quantos QB5
is just over three times smaller; also, the total sample volume has
been rounded up to the next available size of volumetric flask
(50ml).
For example, Table 3 illustrates the preparation of three
concentrations of solution manually (volumetrically) compared to
automatically (gravimetrically) using either (a) an XP205 balance
and an 50mL or 100mL volu-metric flask; (b) a QB3 automated sample
preparation system; (c) a QB5 automated sample preparation sys-tem.
The amounts of substance and solvent consumed for each method are
listing along with the savings.
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Manual (XP205) Gravimetric (QB3) Gravimetric (QB5) Gravimetric
(QX7)
Typical minimum weight 1) 14 mg 10 mg 6 mg 1.4 mg
Recommended safety factor 2) 2 1.5 1.5 1.5
Concentration prepared (mg/mL) 0.5 1.0 1.5 0.5 1.0 1.5 0.5 1.0
1.5 0.5 1.0 1.5
Amount of solid dispensed (mg) 50 50 37.5 15 15 15 9 9 9 2.1 2.1
2.1
Amount of diluent required (mL) 100 50 25 30 15 7.5 18 9 4.5 4.2
2.1 1.05
Substance savings compared to Manual method: 70% 70% 60% 82% 82%
76% 96% 96% 94%
1) Note: The values reported in this table are TYPICAL values
(typical specifications). Determination of the minimum weight on
site will provide the user with the ultimate capability on the
instrument in its specific location.
2) Note: The safety factor quoted is that recommended for stable
environments and trained operators. For unstable environmental
conditions or insufficiently trained operators higher safety
factors should be used.
Table 3: Substance saving based on automated sample preparation
methods
So, if a 1.0 mg/mL solution is prepared manually in a volumetric
flask, 50 mL of solvent and 50 mg of substance are consumed. When
the solution is prepared using an automated gravimetric method on
the QB5 system, 9 mL of solvent and 9 mg of substance are
sufficient (assuming a solvent density of 1 g/mL). A saving of 82%
in both substance and solvent can be realized whilst remaining
compliant with USP General Chapter . Using the QX7 system, the
gravimetric sample preparation method delivers solvent and
substance savings of 96%.
5.6 Volumetric vs. gravimetric comparison
To directly compare the manual volumetric and the automated
gravimetric methods, lets look at a simple prepa-ration comparing
the two techniques. If the method requires a 0.25 mg/mL
concentration then using a volumet-ric system, one would use a 200
mL volumetric flask and weigh out 50 mg of material. Table 4 shows
some of the determinate and indeterminate errors that may be found
in this simple procedure.
Step Volumetric GravimetricDeterminate Errors Indeterminate
Errors Determinate Errors Indeterminate Errors
200 mL container 0.05% Uncalibrated N/A volumetric flasks are
not required
Weigh 50 mg sample 0.1% balanceOthers are accounted
for using a safety factor of 2 or higher
0.1% balanceOthers are accounted
for using a safety factor of 1.5, if automated (in a controlled
lab
environment)
Sample transfer Re-weighing container Powder transfer N/A
transfer of sample to volumetric flask is not required
Fill to mark Reading meniscus and temperature effects
N/A no subjective reading of meniscus required with gravimetric
method
Table 4: Comparing errors in volumetric and gravimetric
processes
As you can see in Table 4, with gravimetric sample preparation
the number of determinate errors is reduced and the indeterminate
errors which tend to be much larger than the determinate ones are
essentially eliminated or accounted for.
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5.6.1 Example of a standard preparation via automated vs. manual
method The data in Table 5 describes an experiment carried out by a
pharmaceutical company who were evaluating gravimetric standard
preparation for an SOP. Using the automated system they wanted to
make 16.5 g total amount of standard at a concentration of 0.603
mg/g. Nine replicates of minimum 10 mg of API were dispensed into
20ml brown glass vials. Following the solid dispense the solvent
(an 80:20 acetonitrile: water mixture) was added gravimetrically to
achieve the target concentration based on the exact weight of API
dispensed. The suspension was dissolved and then a 2 l sample was
injected into the HPLC. In addition, injector precision and
variability in the analytical instrumentation was determined by
comparison of 10 repeat injections of the same solution solution
1.
# Solution Dose API(mg)
Solvent added (g)
Solution Concentration
(mg/g)
Area Area correlated to
0.6 mg/g
Injector Accuracy Solution 1
Area
1 10.105 16.7481 0.60299 2596.8833 2584.00634 Inj. 1
2593.06665
2 10.320 17.1048 0.60298 2595.35474 2582.52818 Inj. 2
2604.43384
3 10.140 16.8063 0.60298 2597.53027 2584.69296 Inj. 3
2604.12378
4 10.125 16.7815 0.60298 2595.5564 2582.72885 Inj. 4
2604.89429
5 10.250 16.9885 0.60299 2597.12427 2584.24611 Inj. 5
2607.99683
6 10.200 16.9058 0.60298 2596.04517 2583.2152 Inj. 6
2605.37231
7 10.130 16.7895 0.60299 2602.42212 2589.51769 Inj. 7
2602.29541
8 10.040 16.6408 0.60297 2609.64868 2596.79455 Inj. 8
2608.33862
9 10.275 17.0297 0.60299 2604.74341 2591.82747 Inj. 9
2593.23218
Inj. 10 2601.73145
Mean 10.176 16.866 0.603 2599.479 2586.617 Mean 2602.549
0.09092503 0.150659114 7.07107E-06 5.000822667 4.983629696
5.37642343
RSD (%) 0.894 0.893 0.001 0.19 0.19 RSD (%) 0.21
Table 5: Automated gravimetric sample preparation data
For the nine individual samples, the weight of solid dispensed
is shown in column 2. The weight of solvent dis-pensed is shown in
column 3 and the concentrations achieved are shown in column 4.
Accurate solvent weigh-ing compensates for any variation in the
amount of solid dispensed (which is already within a narrow range
10.040 and 10.320 mg), to achieve an accurate concentration of
0.603 mg/g in every case. The repeatability of the concentrations
is excellent, as indicated by the low standard deviation, RSD =
0.001% (pale blue highlighted cell). When the peak area is
correlated after analysis, the RSD across these nine solutions is
0.19% (dark blue highlighted cell).
The two columns at the right hand side of Table 5 show ten
injections of the same solution, with the aim of eliminating the
contribution of the analytical system to the overall variability.
The RSD of these samples is 0.21% (green highlighted cell). So, in
conclusion, automated standard preparation with solvent weighing
shows no sig-nificant variability in the solution concentrations,
that cant be attributed to the analytical instrumentation
itself.
When the same experiment was repeated by preparing the
individual standards manually, the results in table 6 were
obtained. The RSD on the correlated areas was 0.57% (compared to
0.19% for automated). This IS signifi-cant compared to the accuracy
of the analytical instrumentation, and means that the variability
can be almost only attributed to the manual preparation process. As
the weighing process and the HPLC analysis are indepen-dent from
each other, the associated %RSD are not added arithmetically but
quadratically in order to determine the %RSD of the peak area. As
the %RSD of the peak area is 0.57% (sample weighed manually), and
the %RSD for the overall variability of the analytical
instrumentation is 0.21%, the calculated %RSD for the manual sample
preparation is 0.54%, which fits nicely to the experimentally
determined %RSD of 0.60% of the manual weigh-ing process. In other
words, for this experiment the limiting factor for the overall
accuracy of the HPLC analysis was the manual sample preparation,
whereas for automated sample preparation the limiting factor for
the overall accuracy is the HPLC itself.
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Manual Dose# Blend dosed
mgFlask Size
mlConc. achieved
mg/ml
1 12.920 20 0.646
2 12.960 20 0.648
3 12.860 20 0.643
4 12.840 20 0.642
5 13.060 20 0.653
6 12.980 20 0.649
7 12.860 20 0.643
8 12.800 20 0.640
9 12.860 20 0.643
10 12.920 20 0.646
Mean 12.906 20 0.645
0.078 0.0 0.004
% RSD 0.60% 0.0 0.57%
Table 6: Manual (volumetric) sample preparation
5.6.2 Summary of Volumetric vs. Gravimetric considerationsThere
are several key elements to consider in the comparison between
manual volumetric and automated gravimetric methods of sample
preparation. These elements are summarized in Table 7:
Manual Volumetric Automated Gravimetric
Substance saving Uses far more substance than neces-sary due to:
Higher minimum weight Higher safety factor Scaling up to
appropriate size of
volumetric flask
Smaller minimum weight = smaller sample sizes
Dispense directly into target vessel and avoid loss on
transfer
No sample spillage or overshooting target weight
Eliminates need to prepare duplicate samples due to OOS weighing
errors
Diluent addition accuracy Manual volumetric method has many
potential error risks: Meniscus reading is subjective Temperature
deviation of contents
compared to calibrated temperature Cross-contamination risk
Incorrect pour/drain times Dishwasher damage Up to 50% failure
rates to comply
with Class A specifications Recalibration recommended every
10 years
Automated gravimetric method is highly accurate: Amount of
liquid needed is calcu-
lated automatically based on the actual weight of solid
which:
Eliminates manual calculation errors Eliminates human error in
choice of
pipette Eliminates human error in choice of
flask The amount of solvent dispensed
gravimetrically is far above the mini-mum weight therefore
measurement uncertainty of this step is negligible
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aper Manual Volumetric Automated Gravimetric
Process and data security Requires hand transcription Relies on
diligence of analyst
Automated data transcription Labels/documentation generated
automatically Integrated RFID chip stores and
tracks substance information and eliminates risk of sample
confusion
Data is electronically recorded and fully traceable
Potential for cross-contamination Volumetric flasks are re-used.
They have to be rinsed before and after each use.
Disposable dosing head is used for a single substance only which
elimi-nates cross contamination
The dosing head is stored in a trans-port container which
protects it from the atmosphere
Each solvent has a unique liquid dosing head. There are no
valves or washing of lines is necessary.
Disposable vial used to prepare samples.
User Safety Manual weighing of solids and sample preparation
involves close contact for user.
Automated dispensing reduces user exposure: The powder is
contained in the
dosing head The dosing head is a closed system Only the required
amount of powder
is released from the dosing head An optional safety enclosure is
avail-
able (QSE2)
Table 7: Comparison of volumetric vs. gravimetric methods
6 Conclusion
The most important measure to guarantee accurate weighing and
consequently to avoid the possibility of OOS due to weighing is the
determination of the minimum weight of the balances. Consequently,
it is impor-tant to always weigh above the minimum weight in order
to comply with the respective accuracy requirements. For automated
dosing systems such as Quantos, the minimum weight is significantly
smaller as compared to manual weighing. It is good practice to
apply a safety factor in order to compensate the variability of the
mini-mum weight due to different operators and changing
environmental conditions, however, the safety factor can be chosen
significantly smaller for automated weighing systems as
environmental effects are reduced and the vari-ability introduced
by the operator is completely removed.
Reducing the occurrence of OOS results in the laboratory
requires close attention to the details of where errors can occur,
a critical evaluation of the overall process workflow, and a
concerted effort to change those practices that lead to OOS results
or errors in the data. New technologies must be brought into the
laboratory to finally improve the data quality that is being
generated by laboratories around the world. In addition, most
companies want and need to achieve higher productivity with the
same or less resources. This efficiency cannot occur with-out a
fundamental change in the way sample preparation is currently
performed which has had little improve-ment for the best part of a
century and still accounts for more than 60% of our time spent in
the laboratory.
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19White Paper METTLER TOLEDO
Gravimetric sample preparation, which is defined as automated
weighing and dispensing of both the solid and the solvent, is based
on the weighing standard GWP. Furthermore, it has several important
benefits in a mod-ern laboratory: it improves user safety due to
reduced exposure risk; it improves process safety via electronic
recording and tracking of data; and it can save substance and
solvent due to reduced minimum weight and elimination of volumetric
flasks.
However, the most important benefit of gravimetric sample
preparation is that it is an innovative way to enhance the accuracy
and drastically reduce the variability in sample processing steps
which has been shown to be the major source of Out-of-specification
results. Some areas of high error risk in the process, such as
those involv-ing use of volumetric flasks, are eliminated
completely. Gravimetric sample preparation has the effect of
reducing laboratory errors and increasing laboratory
efficiency.
7 References
1. United States of America v. Barr Laboratories, Inc., 812 F
Supp 458 (DNJ 1993).
2. U.S. Food and Drug Administration. Guidance for Industry:
Investigating out-of-specification (OOS) test results for
pharmaceutical production. FDA. Available at:
www.fda.gov/downloads/Drugs/GuidanceCompli-anceRegulatoryInformation/Guidances/UCM070287.pdf.
3. Majors, R.E. LC/GC Magazine, 1991, 1997, 2002
4. General Chapter Weights and Balances, US Pharmacopeia USP34
NF29, Rockville, Maryland, 2013, Online-Edition
5. GWP - Good Weighing Practice A Risk-Based Approach to Select
and Test Weighing Instruments, White Paper, Mettler-Toledo AG,
Greifensee, Switzerland, July 2009.
6. Reichmuth A., Fritsch K., Good Weighing Practices in the
Pharmaceutical Industry Risk-Based Qualifica-tion and Life Cycle
Management of Weighing Systems, Pharmaceutical Engineering, Volume
29, Number 6, ISPE, Tampa FL, USA, 2009.
7. Fritsch K., Quenot J.-L., Good Weighing Practices Avoid OOS
Results with Proper Weighing, Pharmaceuti-cal Formulation &
Quality, Volume 14, Number 1, Wiley-Blackwell, Hoboken, NJ, USA,
2012.
8. Evaluation of Measurement Data Guide to the Expression of
Uncertainty in Measurement (GUM), JCGM 100:2008, Bureau
International des Poids et Mesures, Svres, France, 2008. Available
at www.bipm.org.
9. Guidelines on the Calibration of Non-Automatic Weighing
Systems, EURAMET cg-18, Version 3.0, European Association of
National Metrology Institutes, Braunschweig, Germany, 2011.
Available at www.euramet.org.
10. General Chapter "Weighing on an Analytical Balance", Second
Supplement to USP36-NF31, June 2013, Rockville, Maryland,
Online-Edition.
11. Important Technical Guidance on Glassware, Tom Coleman and
Georgia Harris, NIST, Aug. 2005.
12. "Traceability: Volumetric apparatus", LAB15 Guidance,
Edition 2, UKAS, 2009. Available at:
http://www.ukas.com/library/Technical-Information/Pubs-Technical-Articles/Pubs-List/LAB15.pdf
13. Dr Charles Ray, former Associate Director of Analytical
R&D at Bristol-Myers Squibb Co., personal experience from
managing Analytical R&D teams in leading pharma companies.
14. Fritsch, K., Ratcliff, J., Ray Ch., Reducing Variability and
Out-of-Specification Results by Implementing High Quality
Gravimetric Sample Preparation (GSP), Pharmaceutical Engineering,
Volume 32, Number 1, ISPE, Tampa FL, USA, 2012.
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