Life Science Weighing Guide - Mettler Toledo · Life Science Weighing Guide ... Retesting According to SOP or Protocol ... – CAPAs – generated by OOS investigations

Life

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A Practical Guide for Life ScientistsSample and Solution Preparation

Sample andstandard preparation,Gravimetric dilutions,

Buffer preparations

2METTLER TOLEDO Life Science Weighing Guide

3

Content

1. Introduction 4

2. Sources and Impact of Out-of-Specification Results 5

2.1. Out-of-Specification Results 5 2.2. Corrective and Preventive Actions 6

3. Sample Preparation 7 3.1. Volumetric Sample Preparation 7 3.2. Gravimetric Sample Preparation 8

4. Solution Preparation and Dilutions 10

5. Data Management and Traceability 12

6. Summary 13

7. Our Solutions 14

8. References 15

Cont

ent

METTLER TOLEDO Life Science Weighing Guide


1. Introduction

Today’s laboratories are engaged in research, production and quality assurance programs in many different

fields – including pharma, biotechnology and academia – analyzing a broad variety of sample matrices with

different properties. Sample and solution preparation is a crucial part of this process, laying at the heart of

accurate, precise data generation. Inaccurate sample and solution preparation has many consequences, particu-

larly in a regulated environment subject to rigorous quality control regulations, where any non-compliance

may require time-consuming and labor-intensive correction. Repeat analyses may be necessary, consuming

additional precious – often expensive – materials, and confidence in the laboratory data will fall.

Generally, samples for analysis – for example, by HPLC – need to be in solution, and this requires accurate,

precise preparation of samples, standards and reagents. For instance, a laboratory wishing to determine the

amount of an active pharmaceutical ingredient in a tablet must first grind the tablet into a powder, then weigh out

a specified amount for dissolution in a solvent. Standards of known concentrations must be accurately prepared

for instrument calibration, as well as any other solutions or reagents, such as buffers, that are required for the

analytical protocol.

Traditionally, laboratories rely on volumetric methods for sample and solution preparation; however, these manual

techniques have an inherent potential for errors. A newer, more accurate approach – gravimetric sample prepara-

tion – has recently been introduced into the laboratory, minimizing or even eliminating many of the issues associ-

ated with volumetric techniques. This booklet discusses the advantages and limitations of the two methodologies,

and explains how laboratories can benefit from the adoption of gravimetric sample preparation techniques.

Intro

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ion


Sour

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Resu

lts 2. Sources and Impact of Out-of-Specification ResultsAll laboratories, whatever their field of work, must generate reliable, accurate data. This may or may not be

regulation driven, and the impact of an OOS results will vary accordingly. In research laboratories, for example,

the key driver will be the need for method reproducibility and accurate results, while production, testing and

QC laboratories are likely to be strictly regulated, in compliance with stringent GMP/GLP regulations. However, in

striving to ensure highly accurate experimental data, the general principles applying to Out-of-Specification results

are equally relevant to both regulated and non-regulated laboratories.

2.1. Out-of-Specification Results

In the pharmaceutical industry, Out-of-Specification (OOS) results have had a significant impact for many years,

particularly since the 1993 FDA vs Barr Laboratories court ruling. This upheld the Company’s view that an OOS re-

sult does not necessarily constitute a batch failure [United States of America v. Barr Laboratories, Inc., 812 F Supp

458 (DNJ 1993)], and that other potential causes such as a laboratory error should be investigated. However, the

court did not approve of the way Barr was conducting its investigations and, as a consequence, in October 2006

[U.S. Food and Drug Administration. (2006). Guidance for Industry: Investigating Out-of-Specification (OOS) Test

Results for Pharmaceutical Production] the FDA updated its guidelines on the handling and investigation of OOS

results. Since then, the FDA has issued a significant number of observations – 483 – concerning poor investiga-

tions, suggesting that the incidence of laboratory errors may not be as rare as it should be. Unfortunately, there

is no published data showing that for every OOS result generated there were many more minor errors that did

not lead to an OOS result. Such errors may have been classified as a ‘Note to Record’, or simply written in the

laboratory notebook. Often, these errors are not investigated, even though they are probably symptoms of poten-

tially more serious issues with the analytical protocol. LCGC magazine published an article [Majors, R.E. (2002).

Trends in sample preparation. 20 (12), 1098–1113] focusing on the sources of OOS errors and the most time-

consuming activities in an analytical workflow. The survey results indicated that the two main sources of OOS

results are sample processing followed by human error, with sample processing also the most time-consuming

task (Figure 1).

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 (right) and time spent on different activities in the laboratory.


Sour

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lts Even though no follow-up survey has been published, these results still appear valid today. Instrumentation, data

systems and chromatography columns have improved significantly over the last 10 years, providing considerable

time savings in the analytical workflow, yet sample processing is essentially unchanged. It seems fair to assume

that sample processing probably now accounts for even more than 61 % of staff time.

When an OOS result is generated, a great deal of effort is expended on an investigation (Figure 2); obvious OOS

results might be resolved in days, but more serious problems may involve months of work, with the cost running

into many thousands, or tens of thousands, of dollars. Given the large impact of laboratory errors, the best course

of action – whether the laboratory is regulated or not – is to make every effort to avoid them in the first place.

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

Figure 2: The formal process of investigating OOS results.

2.2. Corrective and Preventive Actions

Another significant issue for regulated laboratories is the mounting number of Corrective and Preventative Actions

– CAPAs – generated by OOS investigations. CAPAs typically cause procedural changes to Standard Operating

Procedures (SOPs) and other documents and, over time, become unmanageable and difficult to follow. This has

the potential to create even more issues. The overriding problem with a CAPA is the tendency to assume that it is

an isolated incident, addressing only a specific step in the workflow or blaming a single employee or a simple

laboratory error. While this simple error may be the only issue that needs addressing, frequently the whole pro-

cess needs refining. This applies equally to both regulated and non-regulated laboratories, and is especially

true for sample preparation.


Sam

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ion 3. Sample Preparation

Astonishingly, while enhanced instrumentation and software have provided dramatic improvements in sample

analysis and data processing, sample preparation techniques have changed very little during the last century,

with laboratories continuing to rely on traditional volumetric methods. More recently, a gravimetric approach

to sample preparation has been introduced, offering greater accuracy with less uncertainty of measurement.

3.1. Volumetric Sample Preparation

In volumetric sample preparation, the sample is typically weighed directly into a volumetric flask and the flask

is then filled with solvent. Alternatively, weighing papers may be used, subsequently transferring the sample to a

volumetric flask for dissolution. Frequently, results are handwritten in a laboratory journal, with a risk of transcrip-

tion errors, and any errors are hard to identify and correct. Any variation in the sample preparation process has

the potential to corrupt all downstream measurements, making it difficult to guarantee the accuracy and precision

of the final concentration.

Even with professional handling, the manual volumet-

ric sample preparation process can be cumbersome,

time-consuming, inaccurate and even hazardous, as

well as being prone to day-to-day and operator-to-op-

erator variability. Weighing an exact amount into a vol-

umetric flask is always a challenge, as it is easy to

over or under weigh the sample and, while the use of

weighing papers or boats might initially appear to be a

more straightforward alternative, this is prone to sam-

ple loss during transfer. Operators may forget to wear

gloves, leaving fingerprints – which are very hygro-

scopic – on the flask, and the transfer of body heat

may introduce temperature changes, affecting weigh-

ing accuracy. Electrostatic charge originating from the

operator is also a potential source of weighing inaccu-

racies; clothing can induce a strong attractive charge

on the material on the weighing pan, causing very er-

ratic readings. Weighing errors can be minimized by

preparing larger volumes of sample solution – it is

easier to accurately weigh out 10 mg of compound

manually than 1 mg – but this consumes additional precious, often limited sample and incurs further expense.

Addition of the diluent by volumetric dosing introduces a multitude of indeterminate handling errors, such as read-

ing the meniscus incorrectly and over or under filling the flask, or using glassware at temperatures where thermal

Figure 3: Manual volumetric sample preparation process.


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ion expansion causes the error limit to be exceeded. Using the correct size volumetric flask is vital, as inadvertently

using a larger or smaller volume container than intended will introduce concentration errors. Moreover, as with

any reusable glassware, it is crucial that volumetric flasks are cleaned thoroughly before use to eliminate the risk

of cross-contamination.

Solutions are available to help eliminate the introduction of manual weighing errors. Modern balances equipped

with a hanging weighing pan1 are not affected by air turbulence in the weighing chamber to the same degree as

conventional weighing pans, and micro draft shields2 can also help to minimize air turbulence. Specially designed

holders are available to securely position a variety of weighing vessels for direct dosing without the need for man-

ual handling3, while antistatic weighing funnels4 help reduce inaccuracies due to electrostatic charge, as

well as sample loss during transfer. For completely hands-free operation, weighing balances can be equipped

with optical sensors5.

3.2. Gravimetric Sample Preparation

It is universally accepted that a gravimetric measurement is intrinsically more accurate than a volumetric mea-

surement; in fact, pipettes and other volumetric measuring equipment are calibrated using gravimetric methods.

Gravimetric sample preparation – weighing both the solid and the diluent – is a more recent approach to prepar-

ing samples and solutions. The exact amount of substance dispensed, either manually or using an automated

dosing head, is recorded and used to accurately calculate the amount of solvent to weigh into the container.

Automated liquid dispensing compensates for any over or under weighing by delivering the exact amount of dilu-

ent required to achieve the specified concentration, guaranteeing accurate sample and solution preparation.

Fully automated powder and liquid dispensing systems eliminate the use of weighing papers and volumetric

flasks, helping to further reduce laboratory errors and increase efficiency [Fritsch, K., Ratcliff, J., Ray Ch. (2012).

Pharmaceutical Engineering, 32 (1)].

1 www.mt.com/xpe-analytical2 http://us.mt.com/us/en/home/products/Laboratory_Weighing_Solutions/Accessories/ergoclips/11107869_MinWeigh_Door_micro.html3 www.mt.com/ergoclips4 www.mt.com/smartprep5 http://ch.mt.com/ch/en/home/products/Laboratory_Weighing_Solutions/Accessories/switches_.html

Figure 4: Gravimetric sample preparation.


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ion Automated gravimetric solution preparation offers several advantages compared to volumetric methods. The lower

minimum weight achievable with automated dispensing enables smaller amounts of compound to be accurately

dispensed into any target container, saving material. With laboratories no longer constrained by the availability

of appropriate volumetric glassware, the exact amount of solution required can be prepared; the need to round

up the volume prepared to the nearest flask size is eliminated. This allows consumption of both compound and

solvent to be downscaled significantly, with associated cost savings. In addition, weighing liquids at gram levels,

where the measurement uncertainty contribution is negligible, is very accurate. As the amount of diluent is typi-

cally far above the minimum weight limit of the balance, the relative uncertainty of measurement is almost zero.

Finally, the use of automated powder and liquid dispensing systems decreases the risk of laboratory personnel

coming into contact with potentially toxic substances.

Figure 5: Automated gravimetric solution preparation.


Solu

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ion

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Dilu

tions 4. Solution Preparation and Dilutions

Preparing the sample for analysis is just the beginning. The analytical protocol is likely to require other reagents,

solutions and buffers, as well as reference standards to verify instrument performance and establish calibration

curves to determine the exact concentration of the analyte in the sample.

Reference standard preparation is an everyday task in the analytical laboratory; the more stringent the regulations,

the more important this becomes, and the greater the requirement for detailed documentation. Typically, calibra-

tion standards are prepared volumetrically, relying on the use of volumetric flasks and pipettes. A stock solution is

pipetted into a flask and made up to volume, or into an alternative storage vessel and diluted using

a pipette. The accuracy and precision of this process is dependent on the expertise of laboratory personnel.

As well as the potential errors described earlier, pipetting inaccuracies are now a possibility. It is important to en-

sure that:

• laboratorypersonnelaretrained 6 in correct pipetting techniques, and can demonstrate pipetting competence;

• themostappropriatepipettetypesandvolumesizesareused,withthecorrecttips;

• pipettesareregularlytestedtochecktheaccuracyofdispensing,andapreventivemaintenanceandcalibra-

tion schedule established.

Where serial dilution of reference standards or samples is performed – taking an aliquot from one solution and

diluting this volume to prepare the next solution in the series – this can become quite error prone as the number of

dilution steps increases, and raises further questions:

• Wasthecorrectvolumealwaysdispensed?

• Werethereanybubbleinclusionsthatmightimpactontheresults?

• Howwastheprocessdocumented?

Adopting a gravimetric approach to performing dilutions can help address these issues, transferring an aliquot of

the source solution onto a balance and determining its accurate weight before adding the exact amount of dilu-

ent using an automated liquid dosing unit. Not only can errors be omitted, but the flexibility for dilution becomes

much greater. Any dilution can be made – for example 1:12.5 – even for serial dilutions involving multiple steps

(Figure 3). And, with each step measured on a balance and automatically recorded, the process is fully traceable

and the repeatability of the data points is strongly increased.

6 METTLER TOLEDO’s eLearning course Lab Balances: External Influences and Proper Cleaning offers tips and tricks for improved weighing results. Source: www.mt.com/lab-elearning


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Figure 6: Preparation of a dilution series.

Initial concentration [mg/mL]

Precondition•Quantos QA3/QB5 with QLB (liquid unit) •Pipette(s)•Original stock solution•Vials

Dilute: transfer with pipette, fill with liquid unitMultiple steps possible (depending on max. volume)

Dilute: transfer with pipette, fill with liquid unitOne step each

First working concentration[μg/mL]

2nd working concentration[μg/mL]

3rd working concentration[μg/mL]

n-th working concentration[μg/mL]

1:d 1:d

Parameters (method params)•Max. volume [g] (default: 10.0g) •Working steps (n) [–] (default: 8.0) •Dilution factor (d) [–] (default: 2.0)

Result/Postcondition•Accurately prepared working concentrations •Labels

Variables (user input)•Initial concentration [mg/mL] (default: 1.000mg/mL) •Firstworkingconcentration[μg/mL](default:1.000μg/mL)

Constraints•Do not overfill (m(pipette) + m(liquid target)) < m(max)


Dat

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Tra

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ility 5. Data Management and Traceability

To ensure traceability, the weighing data must be carefully recorded. While this can be performed manually,

marked improvements in quality can be achieved through networking and software support, providing greater

convenience, as well as significant increases in reliability and traceability.

Laboratory software systems are available that enable SOPs to be created and administered remotely7. Admin-

istrators may also have the capability to define safety criteria and make changes on any individual balance or

group of balances, eliminating laborious manual programming of each separate system. Balances may be con-

nected to a PC via an RS232 interface or a network connection (WLAN, Ethernet), with all measured values auto-

matically recorded and saved to an SQL or alternative database; different interfaces enable data to be exported to,

for example, a Microsoft Excel® spreadsheet or a LIMS system. As well as offering greater convenience, this direct

connection to a central database simultaneously improves efficiency, reliability and traceability, aiding CFR 21

part 11 compliant data management. There are also options using radiofrequency identification (RFID) technol-

ogy, ensuring secure and efficient data transfer; sample IDs and weights can be wirelessly transferred from the

balance, eliminating transcription errors and inaccuracies due to incorrect sample selection. Similarly, barcode

readers enable automatic sample identification. The capability to instantly perform targeted searches for individual

measurement or calibration values, both now and in the future, is a key asset to the laboratory, making trouble-

shooting a far more straightforward process. cleaned with a tissue (as long as the pH membrane isn’t touched!).

This stronger electrolyte flow also makes the junction ‘self-cleaning’ to a certain extend.

7 www.mt.com/LabX

Figure 7: Data management with LabX PC software.


Sum

mar

y 6. Summary

Laboratories have traditionally relied on volumetric methods for preparing samples, standards and solutions.

This manual process has changed very little over the years, and has considerable potential for the introduction

of handling errors.

Gravimetric sample preparation offers several clear advantages. It enables a specified concentration to be pre-

pared accurately and precisely, reducing sample variability and impacting positively on the number of instances

of Out of-Specification results, with associated time and cost savings. Automated solvent dispensing reduces

concentration variability and removes the possibility of human error, increasing confidence in the laboratory’s

analytical results. In addition, transcription or labelling errors are eliminated, as data is recorded and printed

automatically. The amount of substance and solvent used can also be reduced by up to 90 %, with the added

benefit of greatly reducing the likelihood of exposing laboratory personnel to potent substances.


Our

Sol

utio

ns 7. Our Solutions

METTLER TOLEDO’s instruments support customers in the life science industry, providing the right solution to meet high accuracy, user comfort, measurement reliability and other requirements.

METTLER TOLEDO offers an entire portfolio of solutions for both volumetric and gravimetric sample preparation, gravimetric dilution and data integration. Enhanced with smart accessories, these instruments can meet a broad variety of customer requests from easy operation to fully automated analysis.

Method Solution

Volumetric sample preparation

Analytical balances XPE205 with ErgoClipsReadability 0.01 mgCapacity 220 gTypical minimum sample weight (USP) 21 mgMore models available.

Gravimetric sample preparation and dilution

Quantos QA3 systemReadability 0.01 mgCapacity 220 gAutomated liquid dispensing yesTypical minimum sample weight (USP) 21 mgMore models available.

Automated standard and sample preparation

Quantos QB5 systemReadability 0.005 mgCapacity 220 gAutomated powder dispensing yesAutomated liquid dispensing yesTypical minimum sample weight (USP) 14 mgMore models available.

Workflow integration Quantos WorkflowBoxAllows full workflow and data integrationBased on LabX software solution.


Refe

renc

es 8. References

1. United States of America v. Barr Laboratories, Inc., 812 F Supp 458 (DNJ 1993).

2. U.S. Food and Drug Administration. (2006). Guidance for Industry: Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production. Available at: www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070287.pdf

3. Majors, R.E. (2002). Trends in sample preparation. LCGC North America, 20 (12), 1098–1113.

4. Fritsch, K., Ratcliff, J., Ray Ch. (2012). Reducing Variability and Out-of-Specification Results by Implementing High Quality Gravimetric Sample Preparation (GSP). Pharmaceutical Engineering, 32 (1), ISPE, Tampa FL, USA.

For more information

Mettler-Toledo International Inc.CH-8606 Greifensee, SwitzerlandTel. +41 44 944 22 11Fax +41 44 944 30 60

Subject to technical changes© 03/2014 Mettler-Toledo AGGlobal MarCom Switzerland

www.mt.com/xpe-analytical

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Life Science Weighing Guide - Mettler Toledo · Life Science Weighing Guide ... Retesting According to SOP or Protocol ... – CAPAs – generated by OOS investigations