U.S. patent application number 09/730126 was filed with the patent office on 2002-08-08 for automated test protocol.
Invention is credited to Andrews, Richard Wayne, Corbin, Virginia L..
Application Number | 20020107652 09/730126 |
Document ID | / |
Family ID | 24934021 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020107652 |
Kind Code |
A1 |
Andrews, Richard Wayne ; et
al. |
August 8, 2002 |
Automated test protocol
Abstract
A method and apparatus for automating the qualification process
for chromatographic systems. Automation technology and regression
analysis are used for qualifying a chromatography system. The
trained operator prepares the chromatography system to ensure that
the samples, solvents, and the separation column are ready for
analysis. The qualification of the detector, the solvent delivery
system, the sample manager, the gradient proportioning system, the
column heater, and the delay volume of the chromatography system
are completed without the necessity of operator intervention.
Regression analysis is performed to compute performance statistics
that demonstrate the accuracy, linearity, and precision of the
chromatographic system and quantify its suitability for
chromatographic analysis.
Inventors: |
Andrews, Richard Wayne;
(Rehoboth, MA) ; Corbin, Virginia L.; (Medway,
MA) |
Correspondence
Address: |
Anthony J. Janiuk, Esq.
WATERS CORPORATION
34 Maple Street
Milford
MA
01757
US
|
Family ID: |
24934021 |
Appl. No.: |
09/730126 |
Filed: |
December 5, 2000 |
Current U.S.
Class: |
702/104 |
Current CPC
Class: |
G01N 2030/8804 20130101;
G01N 30/88 20130101; G01N 30/02 20130101; G01N 2201/129
20130101 |
Class at
Publication: |
702/104 |
International
Class: |
G01D 018/00 |
Claims
What is claimed:
1. An automated method for qualification of a chromatography
system, having a detector, solvent delivery system, sample manager,
and column, comprising the steps of: preparing the chromatography
system to ensure that samples, solvents, and column are ready for
analysis; qualifying the detector to ensure operation within
specified detection parameters; qualifying the solvent delivery
system to ensure operation within specified solvent delivery
parameters; qualifying the sample manager to ensure operation
within specified sample delivery parameters; utilizing regression
analysis to compute performance of accuracy, linearity, and
precision of the chromatographic system; and validating performance
of the chromatography system based upon said regression
analysis.
2. The automated method for qualification of a chromatography
system according to claim 1, wherein said chromatography system
includes a gradient proportioning system, further including the
step of qualifying the gradient proportioning system to ensure
operation within specific gradient proportioning parameters.
3. The automated method for qualification of a chromatography
system according to claim 1, wherein said chromatography system
includes a delay volume, further including the step of qualifying
delay volume of the chromatography system to ensure operation
within specific delay volume parameters.
4. The automated method for qualification of a chromatography
system according to claim 1, wherein said chromatography system
includes a column heater, further including the step of qualifying
the column heater to ensure operation within specific column
temperature parameters.
5. The method of claim 1, wherein said detector is an absorbance
detector.
6. The method of claim 1, wherein said detector is a photodiode
array detector.
7. The method of claim 1, wherein said detector is a fluorescence
detector.
8. The method of claim 1, wherein said detector is a refractive
index detector.
9. The method of claim 1, wherein said method for qualification is
compliant with FDA regulations relating to electronic signatures
and electronic records.
10. The method of claim 9, further including the step of generating
a report to confirm compliance of the chromatography system.
11. The method of claim 10, further including the step of reviewing
said report to confirm compliance of the chromatography system.
12. The method of claim 10, wherein said report is compliant with
Good Laboratory Practice.
13. The method of claim 10, wherein said report is compliant with
Good Manufacturing Practice.
14. The method of claim 10, wherein said report is compliant with
Good Clinical Practices.
15. The method of claim 10, wherein said report is a hardcopy
report.
16. The method of claim 1, wherein said regression analysis
generates data that is placed within an Oracle database.
17. An automated method for installation qualification of a
chromatography system, comprising the steps of: storing details of
installation data within a Oracle database table; creating an
unique sequence for each record stored in said table; preventing
the deletion of said records; and accessing said data using data
objects.
18. An apparatus for use with a computer system, including a
central processing unit, and an application program, for qualifying
a chromatography system comprised of a plurality of components,
each of the plurality of components operating on parameter data and
producing output values therefrom, the apparatus comprising:
storage means controlled by the central processing unit and
cooperating with the computer system to store the application
program; means for storing predicate rules for detecting invalid
data, the predicate rules comprising precondition rules for
detecting invalid parameter data and post condition rules for
detecting invalid output values generated by one of the plurality
of components; means responsive to the stored application program
and to the stored predicate rules for compiling the predicate rules
and the application program to generate an executable program
module, an executable precondition module and an executable post
condition module in a common library; and means controlled by the
central processing unit and responsive to the output values for
applying the output values to the post condition module to detect
invalid output data.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to chromatography systems, and
more particularly the use of automation technology in the
qualification of chromatography systems.
BACKGROUND OF THE INVENTION
[0002] Chromatography systems are used to analyze various products
developed by pharmaceutical companies, hospitals, and government
laboratories. Such products in many cases are regulated by the
United States Food and Drug Administration (the "FDA") and other
foreign regulatory agencies, therefore regulatory guidelines
require the validation of these chromatography systems for
laboratories submitting data, e.g. pharmaceutical samples to the
above regulatory agencies. The regulatory requirements demand that
the chromatography systems that are used to analyze products must
meet certain minimum requirements as many regulatory agencies will
not accept data from laboratories that have not established that
they are using validated chromatography systems.
[0003] When a chromatography system satisfies the validation
requirements, it is said to be "qualified". A qualified
chromatography system generally must meet the articulated standards
in three separate areas. The three areas are installation,
operation, and performance. Each area is described below.
[0004] The installation qualification ("IQ") verifies that the
chromatography system satisfies three conditions associated with
the installation of the system. First, the IQ establishes the
chromatography system is received as designed. Second, it verifies
the chromatography system is installed properly. Lastly, the IQ
verifies that the environment where the system is installed is
appropriate.
[0005] The operational qualification ("OQ") ensures the instruments
which comprise the chromatography system function according to
their individual operational specifications in the chosen
environment. An OQ does not specifically verify that individual
modules successfully perform as part of an integrated system.
[0006] The performance qualification ("PQ") ensures the integrated
chromatography system routinely performs according to
specification.
[0007] Conventional methods for qualifying chromatography systems
include manuals, qualification workbooks, and metrology based
qualifications. An example is the Waters HPLC Systems Qualification
Workbook developed by Waters Corporation of Milford, Mass. This
highly manual and labor intensive process takes from 10-12 hours to
finish per chromatographic system. Qualification via qualification
workbooks is extremely time consuming because the individual
modules and the integrated system are qualified separately.
[0008] Attempts to automate the qualification systems, such as with
the Hewlett Packard 1100, have not been successful. The HP method
is merely a manual system with the only improvement being that the
workbook for the use of the system is contained on a cd-rom.
[0009] Another disadvantage of conventional methods of
qualification is that different samples, solvents, and methods are
used in the qualification of the modules (OQ) and those used for
the qualification of the system (PQ). are different than those used
by the lab on a daily basis. Consequently, a significant amount of
time is lost in removing solvents and samples from the
chromatograph and documenting the multiple reagents and samples
used in system qualification. Therefore, conventional methods take
too much time and require constant technical human intervention.
Because of the demands of continual human intervention, the cost to
industry is excessive. Additionally, the need to maintain and
retrieve the various qualification reports is burdensome and not
amenable to the advantages of electronic format.
SUMMARY OF THE INVENTION
[0010] The present invention provides methods and apparatus for
automating the qualification process for chromatographic
systems.
[0011] According to the invention automation technology and
regression analysis are used for qualifying a chromatography
system. In order to practice the present invention, certain steps
must be performed. The initial step involves preparing the
chromatography system to ensure that the samples, solvents, and the
separation column are ready for analysis. After the chromatography
system has been prepared, automated steps are performed to qualify
the detector, the solvent delivery system, the sample manager, the
gradient proportioning system, the column heater, and the delay
volume of the chromatography system. Regression analysis is
performed to compute performance statistics that demonstrate the
accuracy, linearity, and precision of the chromatographic system
and quantify its suitability for chromatographic analysis.
[0012] In an illustrative embodiment, the automated qualification
systems application is built using the Millennium.sup.32 vV3.20
Toolkit Option (Professional Edition, Waters Corporation) and
Microsoft Visual Basic 6.0 (Enterprise Edition, Microsoft
Corporation).
[0013] Advantages of the invention include the use of automation
technology to provide a substantially faster way to qualify
chromatography systems. Less time is required for qualification,
thus the cost of qualification is lowered enabling more frequent
qualifications. The method according to the invention minimizes
contamination of the chromatography systems with solutions which
are not suitable as mobile phases that could interfere with normal
operation in subsequent analyses. The testing is based on
"normal/intended" use of chromatograph and data system, which is
consistent with the current FDA regulations and does not use
procedures and materials substantially different from the primary
application. Further, the operator, after initial procedures are
performed, is allowed to utilize their time attending to other
matters, as the invention requires no additional human intervention
during the qualification process. The production of various reports
in an electronic format allows off site review and the generation
of varied format reports. Test results can be archived in an
efficient electronic format.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The foregoing and other features and advantages of the
present invention will be more fully understood from the following
detailed description of illustrative embodiments, taken in
conjunction with the accompanying drawings in which:
[0015] FIG. 1 shows a typical chromatography system.
[0016] FIG. 2 shows a flow chart of the steps used to qualify a
chromatography system according to the present invention.
[0017] FIG. 3 shows an illustration of the software components
utilized in the development of the application.
[0018] FIG. 3a shows a typical chromatogram
[0019] FIG. 4 shows an illustration of the response curves for
detectors demonstrating various linear dynamic range values.
[0020] FIG. 5 illustrates that the change in refractive index with
concentration is not necessarily a linear phenomenon.
[0021] FIG. 6 illustrates that the change in refractive index of
sucrose water mixtures is linear over a wide range of
compositions.
[0022] FIG. 7 illustrates a calculation relating the void volume to
flow rate.
[0023] FIG. 8 illustrates injection volume accuracy.
[0024] FIG. 9 illustrates the delay volume as well as the column
void volume and the volume of mobile phase.
[0025] FIG. 10 illustrates the two sets of retention time as a
function of column temperature.
[0026] FIG. 11 illustrates a reasonable degree of linearity that
occurs over a shorter range of temperatures.
DETAILED DESCRIPTION
[0027] As shown in FIG. 1, a conventional chromatography system
typically includes a solvent delivery system 12, a sample manager
10, a column 14, a detector 16, and a Data System 18 The present
invention provides a method of using automation technology for
qualifying chromatography systems as required by the FDA.
[0028] A flow chart as shown in FIG. 2 illustrates the steps of an
illustrative embodiment for performing a qualification according to
the invention. In the illustrative embodiment the automated method
is initiated by launching 101 a Millennium.sup.32 Toolkit. Upon the
launch 101 of the Toolkit, the application retrieves the system
information from an Oracle database 204 (FIG. 3) and creates a
Millennium project and configures 102 the system in accordance with
Millennium as is known in the art. The application is then
configured 102 according to the project selected. The configuration
102 of the application incorporates the control of various
components of the application. The selection of the project type
and the acquisition server are completed within the configuration
102 of the system. The chromatographic system is identified and the
specific features of that system are confirmed. The presence of a
column heater and the type of detector that is to be used for
qualification are selected. The selection of the type of flow cell
contained within the chromatographic system and the different
instrument modules contained within the system are identified
during the configuration 102 step and confirmed. The system is then
validated 103 based upon the configuration 102 step. Based upon the
qualification method selected during the configuration step 102 the
application creates a matrix 104 with a specific sample and method
queue within the Millennium.sup.32 application. The methods that
are selected during the configuration step 102 are specific for the
various qualification methods within the illustrative embodiment.
The matrix determines the specific chemistries and mathematical
algorithms employed within a specific chromatography column.
[0029] Once the configuration 102 of the system has been completed,
the trained operator 100 of the chromatography system prepares 105
the chromatograph for the automated qualification of the
chromatography system. The trained operator 100 during the
preparation step 105 verifies that the samples required for the
entire qualification procedure are in the proper location in the
proper carousels. The preparation 105 entails setting up the
solvent manager, placing standard samples in the sample manager,
and equilibrating the chromatographic column to ensure that the
system is well equilibrated and ready for analysis.
[0030] Once the preparation 105 of the chromatograph is completed,
test injections 106 are run which verify that system preparation
105 is completed correctly. If the test injections 106 are within
the specifications the trained operator 100 queues the running of
the automated qualification process and the additional tests needed
for qualification are then performed automatically without the need
for trained operator 100 intervention. If the test injections 106
are not within specifications the preparation 105 of the
chromatograph is repeated.
[0031] As illustrated in FIG. 2 it is critical that the
qualification of Detector 108 is done first, since most of the
remaining measurements are based on the accuracy and linearity of
the detector. The qualification of a solvent delivery system 109, a
sample manager 110, a gradient proportioning system 111, and the
delay volume 112 are conducted in the illustrative embodiment in a
sequence 120 shown in FIG. 2. This sequence 120 is not critical and
in alternative embodiments the steps can be performed within a
different sequence or performed substantially simultaneously. The
qualification of the Column Heater 113 is optional since not all
systems include this component. The Column Heater qualification 113
requires increasing the column temperature. It must therefore be
done after the sequence 120 shown in FIG. 2. It is critical that
the Column Heater qualification 113 be done after the sequence
since chromatography systems usually have no active cooling
mechanism.
[0032] In conformance with the methods that are created in the
sample and method queue 104, data is collected 114 that is
generated by the automated qualification process and placed within
an Oracle database 204 (FIG. 3). The collected data 114 is
processed 115 and the results are stored within the database 204
(FIG. 3). The Millennium.sup.32 201 (FIG. 3) software then creates
reports 116 in a format that is in conformance with regulatory
requirements. A hard copy 117 of the reports 116 is printed for
review 118 by the trained operator 100 of the chromatography
system. The trained operator 100 confirms either the compliance 119
of the chromatography system or the failure 121 of the
chromatography systems to perform within acceptable standards.
Based on the generated reports 116 any deficiency within the system
is identified and corrective action 122 is performed.
[0033] Referring to FIG. 3 the automation, analysis, and generation
of the above qualification method is accomplished within the
illustrative embodiment by utilizing an add-on application 203 to
the Millennium.sup.32 software 201 (Waters Corporation). This
add-on application 203 is built using the Millennium.sup.32 v.3.20
Toolkit Option 202 (Professional Edition, Waters Corporation) and
Microsoft Visual Basic 6.0 (Enterprise Edition, Microsoft
Corporation). The Millennium.sup.32 version 3.20 Toolkit 202
consists of a Toolkit Server, a Toolkit Extensions Server and the
ActiveX Processing Control. Millenium Toolkit 202 is described in
detail in Toolkit Programmer's Reference Guide, P/N 71500016005,
Revision A available from Waters Corporation Milford, Mass., which
is incorporated herein by reference. The Toolkit 202 is essentially
an Applications Programming Interface (API) that defines the
servers' objects and their methods and properties. Only pre-built
objects available in Visual Basic 6.0 such as the form, the frame
the command button, and the checkbox are used in the creation of
the add-on 203 application. The add-on 203 application utilizes the
Toolkit 202 and the underlying Millenium.sup.32 201 application
running on Windows 98/Windows NT 200.
[0034] The Millennium.sup.32 Toolkit 202 is based upon component
integration technology commercially available in the software
industry, the Microsoft Component Object Model ("COM"). The Toolkit
Option 202 contains over 30 programmable COM objects that allow the
use of development platforms such as Microsoft Visual Basic or
Microsoft Office to create specialty applications that work
interactively with the Millennium.sup.32 201 software. The basic
operation of the Millenium Toolkit 202 is in a wizard format. The
resulting application 203 is compliant with the FDA's Electronic
Records and Signatures Rule (21 CFR Part11), which is a requirement
for any laboratory operation under Good Laboratory Practice ("GLP")
or Good Manufacturing Practice ("GMP") because Millennium.sup.32 is
compliant.
[0035] In order to record details such as IQ data a table is
created in Millennium.sup.32 Oracle database. Criteria associated
with qualification data are the main make up of the table. A
sequence is also created so that each record in the table is given
a unique ID. Each type of record is version controlled and new
versions will be recorded when new work is carried out. The records
are stored in the database table. In this illustrative embodiment
there is no delete functionality available. If there are
discrepancies with the current IQ record then the trained operator
100 can re-write the details and store a new record. Instead of
calling external SQL scripts the application uses the Command
objects of a Data Environment Designer connection object. The data
source is accessed using ActiveX Data Objects (ADO) from an
OLE.RTM. DB provider for Oracle.
[0036] ADO is designed as an easy-to-use application level
interface to Microsoft's data access paradigm, OLE.RTM. DB.
OLE.RTM. DB provides high-performance access to any data source,
including relational and non-relational databases, email and file
systems, text and graphics, custom business objects, and more. ADO
is implemented within the illustrative embodiment for minimal
network traffic in key Internet scenarios, and a minimal number of
layers between the front-end and data source. The above methods
provide a lightweight, high-performance interface. ADO is called
using a familiar metaphor, the OLE.RTM. Automation interface.
[0037] OLE.RTM. DB is a low-level interface that introduces a
"universal" data access paradigm. That is, OLE.RTM. DB is not
restricted to ISAM, Jet or even relational data sources known in
the art, but is capable of dealing with virtually any type of data
regardless of its format or storage method. This versatility means
that data in the illustrative embodiment can be accessed that
resides in an Excel spreadsheet, text files, or even on a mail
server such as Microsoft Exchange.
[0038] In the illustrative embodiment Visual Basic 6.0 is utilized
to increase the flexibility of OLE.RTM. DB through ADO, programmer
interface. Since OLE.RTM. DB is not designed to be accessed
directly from Visual Basic due to its complex interfaces, ADO
encapsulates and exposes virtually all of OLE.RTM. DB's
functionality. Additionally, the Data Environment Designer provides
an interactive, design time environment for creating programmatic
run time data access. The property values are set for the
Connection and the Command objects, write code to respond to ADO
events, execute Commands, and create aggregates and hierarchies.
The Data Environment objects are also placed onto forms or reports
to create data-bound controls.
[0039] The Data Environment designer is used to create a Data
Environment object. The Data Environment object includes Connection
and Command objects, groupings, and aggregates. In designing the
Data Environment the database is identified that contains the
information for the run-time objective of creating a Data
Report.
[0040] To access data using the Data Environment, a Connection
object is created. Every Data Environment includes at least one
Connection object. A Connection object represents a connection to a
remote database that is used as a data source. Upon adding a Data
Environment to the Visual Basic project, the Data Environment
designer automatically includes a new connection, called
Connection1. The configuration of the illustrative embodiment is
such that the Data Environment opens the connection and obtains
metadata from the connection, including database object names,
table structures, and procedure parameters. The source for the data
environment connection is defined using the data link properties
dialog box. In the illustrative embodiment, the Microsoft OLE.RTM.
DB provider for Oracle is the choice.
[0041] The Command objects in this application are based on both
the database table object and Structured Query Language (SQL)
queries. Use is made of the fact that pre formatted Commands to
carry out SQL queries can be revised at run time so that the
changed query variables will cause data retrieval to change.
[0042] The Microsoft Data Report designer is used in conjunction
with the data source of the Data Environment designer, reports are
created from the database qualification table for IQ. In addition
to creating printable reports, one can also export the report to
HTML or text files. As previously indicated query criteria change
so to does the output of the report as the controls on the report
are bound to the changed Command.
[0043] The sample set methods to be chosen depends on the
configuration 102 indicated by the trained operator 100 of the
automated test system. The value properties are examined for the
various qualification and text Checkboxes and the status indicated
on the Configuration Frame. Whether a system is tested for
Operational & Performance Qualification or full System
Qualification there are different sample set methods to be used.
The three variables "Detector Type", "Temperature Control" and
"Cell Type" influence the selection. Hence a three dimensional
sample set matrix/array with the above coordinates and containing
various suffixes is provided within the software. When the suffixes
are concatenated with the chosen tests to be performed the result
is a run sequence that can be passed to the instrument server. As
shown in FIG. 2, a series of test injections 106 is always used as
part of the run as a precursor to successful qualification. Queuing
the sample sets involves the initiation of a Toolkit Instrument
object and using the run method with the names of the sample set
methods. A timer cycle is used to enable the monitoring of Toolkit
Instrument connection status. Each and every time a proposed run is
to be queued the revision number for qualification attempts is
incremented in the database table.
[0044] The increment is also tied to the sample set name that
appears in "Run Samples".
[0045] The following chemistries and mathematical theories of the
illustrative embodiment allow the above software to integrate the
installation, operational and performance qualifications procedures
into an automated system process by developing standards that are
linear and therefore amenable to automated analysis. The
quantitative analysis of a standard chromatogram as shown in FIG.
3a requires the association of a peak area (or height) with the
mass or concentration of the analyte injected via an appropriate
calibration curve constructed from the injections of standards. The
principal parameters associated with a chromatogram are the
retention times 301 and peak areas 302. The general metrics of
instrumental analysis for the reported responses (retention times
301 and peak areas 302 ) are the basis of qualification for both
manual and automated systems. The precision, accuracy, and
linearity of retention times 301 and peak areas 302 are dependant
upon factors discussed below. The detector performance parameters
that are measured in the present invention during the qualification
of the detector which most directly affect the chromatograms are
linear dynamic range and wavelength accuracy. Other detector
performance parameters such as detector dispersion, noise, and
drift are highly method dependent. Therefore, it is not useful to
produce automation for those factors that are not universal in
their impact.
[0046] Referring to FIG. 4, an illustration of the response curves
for detectors demonstrates various linear dynamic range values. The
value associated with each calibration curve is the absorbance at
which the observed value is 95% of the value predicted by Beer's
Law in accordance with an older ASTM protocol. The procedure calls
for the drawing of the curved line which fits the data and the
extrapolation of the initial slope. This is followed by an
estimation of the 5% negative deviation point. The procedure is
highly subjective and not adaptable for an automated test system.
An alternate approach utilized in the qualification of the detector
in the illustrative embodiment, that is amenable to automation,
also relies on Beer's Law.
A=.epsilon.*b*C
[0047] If the measured absorbance is divided by concentration, the
apparent sensitivity is calculated.
S=A/C=.epsilon.*=constant
[0048] The apparent sensitivity, S, is not constant over the
dynamic range of the instrument, but the magnitude of the relative
standard deviation (% RSD) of the sensitivity is a good measure of
its degree of variation. If sensitivities are measured at values of
absorbance which fall on both the linear and curved portions of the
calibration curve, this RSD of the sensitivity becomes a good
measure of linear dynamic range.
[0049] An example of such a calculation is shown hereinafter in
Table 1.
1TABLE 1 Linear Dynamic Range Calculation Based on Sensitivity
Concentration 2.5 AU 2.0 AU 1.6 AU S_2.5 AU S_2.0 AU S_1.6 AU 5
0.499 0.498 0.495 0.0998 0.0995 0.0990 10 0.996 0.990 0.980 0.0996
0.0990 0.0980 15 1.486 1.467 1.436 0.0990 0.0978 0.0957 20 1.955
1.901 1.820 0.0978 0.0950 0.0910 25 2.371 2.241 0.0948 0.0896 30
2.678 0.0893 % RSD 4.21 4.22 3.72 Max AU 2.678 2.241 1.820
[0050] The calculation is illustrated as such for the detector
labeled 2.5 AU and the slope of the calibration curve has a
relative standard deviation of only 4.21% for absorbances
.ltoreq.2.678. The detector labeled 1.6 AU has a relative standard
deviation of 3.72% for absorbance .ltoreq.1.82.
[0051] This approach does not require non-linear curve fitting like
prior manual methods illustrated in FIG. 4 and generates
numerically simple results, that are used in the illustrative
embodiment for the qualification of the detector 108 that is
adaptable for the automated system of the present invention
described herein before. The selection of specific target
absorbance values and setting of control values for the %RSD
observed for a specific detector model can be done in a
straightforward manner, if non-linearity is modeled as stray
light.
A.sub.meas=log.sub.10{(1+%s/100)/(10.sup.-A+%s/100)}
[0052] where A.sub.meas=the measured absorbance, A=true absorbance
in absence of stray light, and %s=the stray light expressed as a
percent.
[0053] By setting A.sub.meas=0.95 A, the apparent stray light for a
given value of linear dynamic range (expressed as absorbance at
which there is a 5% deviation from linearity) can be calculated.
Thus, the control value for %RSD of the apparent sensitivity can be
directly correlated to the design specification for a specific
detector.
[0054] The choice of a probe compound in the present invention is
based on ensuring that the boundary conditions for Beer's Law are
met. In the illustrative embodiment the above conditions are:
spectral bandpass is small relative to peak width, dilute solution,
constant refractive index, and simple chemical equilibrium. In this
illustrative embodiment, caffeine dissolved in water:methanol
mixture and measured at 272 nm meets all of the criteria and is
stable and available in high purity.
[0055] The linear dynamic range of a detector is best measured at
.lambda..sub.max. This ensures that wavelength accuracy errors will
not contribute to the measurement of linear dynamic range and that
the spectral bandpass (slit width) of the detector will have
minimum impact on the measurement. This is consistent with good
spectroscopic practice. To determine the linear dynamic range of a
detector, a series of samples which generate chromatographic peaks
with heights ranging from 0.1 to 2.2 AU is injected into the
chromatography system. The probe compound used for wavelength
accuracy is also used for the measurement of linear dynamic range.
This reduces the number of different samples and solvents required
for qualification and ensures that the linear dynamic range is
measured at .lambda..sub.max.
[0056] In order to verify the wavelength accuracy of the detector
during the qualification of the detector 108 of the present
invention, a probe compound must be selected. Suitable probe
compounds must not react with the column or any of the solvents. It
also should have well defined characteristics that can be traced
back to known (well characterized) standards. Most importantly, the
UV spectrum of the probe compound must have at least two well
resolved absorbance peaks which should be in the primary wavelength
range of absorbance detectors (200-400 nm).
[0057] Table 2 shown below summarizes several probe compounds which
are helpfuil in measuring wavelength accuracy in HPLC
detectors.
2TABLE 2 Wavelength Accuracy Probe Compounds Name Solvent
Wavelength(s) Note(s) Erbium (III) Perchlorate Water 255, 379, and
523 nm NIST and ASTM traceable; multiple (all peaks are highly
symmetric) wavelengths over wide range; simple chemistry. Caffeine
Water:methanol 205 and 272 nm Two points covering primary
wavelength range; easily automated. Uracil Methanol 257 nm Highly
symmetric absorbance peak; compatible with detectors having larger
spectral bandpass. Anthracene Water: 252 nm Highly asymmetric peak
at 252 nm; acceptable Acetonitrile choice for detectors with small
(.ltoreq.5 nm) spectral bandpass Holmium oxide Perchloric acid
Multiple asymmetric peaks NIST traceable; best for detectors with
cuvette (10% in water) accessory; requires very narrow (.ltoreq.2
nm) bandpass
[0058] The first two compounds are the favored choices for
wavelength accuracy measurements. Uracil is suitable for wavelength
accuracy measurements for older detectors which have larger
spectral bandpass values.
[0059] One suitable probe compound is a solution of caffeine
dissolved in methanol and water. AUV spectrum of the caffeine,
methanol, and water solution has principal absorbance peaks at 205
nm and 272 mn. The observed wavelength of maximum absorbance
(lambda max, .lambda..sub.max) must match the reference values
within the detector specifications, typically .+-.1.5 to 2 nm.
[0060] In the OQ of photodiode array detectors (PDA), which acquire
an absorbance spectrum rather than the primary single wavelength
data that conventional absorbance detectors acquire, the OQ of the
detector remains the same. The wavelength accuracy and linear
dynamic range are the variables that either a manual qualification
or as in the present invention an automated qualification need to
access.
[0061] Measurement of the linear dynamic range of a PDA detector
ensures that both stray light and resolution are unchanged from the
design parameters of the detector.
[0062] The OQ of fluorescence detectors, in the automated process
of the illustrative embodiment present invention, is accomplished
by the principles set forth below. Fluorescence detectors operate
by irradiating the sample with light at an excitation wavelength
(.lambda..sub.ex) and measuring the intensity of the light emitted
at the emission wavelength (.lambda..sub.em). The relationship
between concentration and the observed intensity of emission is
given by Equation 1.
Fluorescence=F=.function.(.theta.)*g(.lambda..sub.em)*.phi..sub..function.-
*P.sub.o(.lambda..sub.ex)* (1-exp{.epsilon.bC}) Equation 1:
[0063] Where .function.(.theta.) is the geometric collection
efficiency of the detector, g(.lambda..sub.em) is the
photomultiplier's response at the emission wavelength,
.phi..sub..function.is the quantum efficiency of the analyte,
P.sub.o(.lambda..sub.ex) is the radiant power of the source at the
excitation wavelength, .epsilon. is the molar absorbtivity, b is
the path length, and C is the concentration of the analyte.
[0064] Equation 1 can be simplified by combining constants and
expanding the exponential term in a Taylor series to give Equation
2.
Fluorescence=F.congruent.constant*P.sub.o|l (.lambda..sub.ex)*C
Equation 2:
[0065] This equation is correct only for small values of absorbance
(<0.01) where the second order and higher terms of the Taylor
series can be ignored. Examination of Equations 1 and 2 indicate
that the following parameters will strongly influence the observed
fluorescence signal. These parameters are as follows: excitation
wavelength, emission wavelength, source intensity, the excitation
wavelength, response characteristics of the photomultiplier tube,
concentration of the analyte, and the quantum efficiency of the
analyte.
[0066] The first four factors are directly coupled to the detector
while the last two factors are strongly assay dependent. The
suitability of a fluorescence detector to perform chromatographic
analysis can be determined by confirming wavelength accuracy for
both the excitation and emission monochrometers and confirming that
the signal to noise of a stable luminescence process meets an
acceptance criteria. Because the absolute magnitude of the
fluorescence signal is directly proportional to the source
intensity which will vary as the lamp ages, it is not possible to
benchmark performance to the peak height under specific gain
conditions. A more measurable approach for the determination of the
condition of the detector is to measure the signal to noise ratio
for a well-known luminescence signal.
[0067] The Raman shift of water provides a convenient test probe
for fluorescence detectors. When water is irradiated with light,
most water molecules scatter the light elastically, i.e., with no
transfer of energy. A small fraction of the water molecules absorb
sufficient energy and re-emit the photon at a lower frequency. If
the excitation light is at 350 nm, the Raman scattering is observed
at 397 nm. The signal to noise ratio of the emission peak at 397 nm
and its location at 397 nm confirm the suitability of the detector
with respect to response and wavelength accuracy. The observed
signal to noise ratio for the Raman band of water will reflect the
source intensity, the purity of the water used for the measurements
(HPLC grade water is appropriate), and the signal processing
(including filtering, spectral bandpass of both monochrometers, and
the illuminated volume of the flow cell). The Raman signal to noise
ratio should meet or exceed the manufacturers' specification. If
either the excitation or emission monochrometer's wavelength
accuracy can be independently verified, the relationship between
the observed .lambda..sub.max of the Raman band (397 nm with 350 nm
excitation) can be used to verify the remaining monochrometer. If
the flow cell is removed and the photomultiplier gain is kept
small, the presence of Hg emission lines in the light from
fluorescent room lights can be used to verify the wavelength
accuracy of the emission monochrometer.
[0068] Verification of linear dynamic range for a fluorescence
detector is not required because Equation 1 is inherently
non-linear and behaves linearly only under specific conditions. Two
additional phenomena--the so-called "inner filter" effect and
self-absorbtion--add additional exponential terms to Equation 1.
The inner filter effect occurs when the sample (or its solvent)
absorbs a portion of the excitation energy in a portion of the flow
cell which is not imaged onto the emission monochrometer. The
result is that the radiant energy is decreased without a concurrent
increase in the measured emission intensity. If the absorbance
spectnrum overlaps the emission spectrum, some of the photons
emitted by the analyte molecules will not be observed. The
magnitude of both of these effects is dependent upon the analyte
concentration.
[0069] Consequently, the linear dynamic range of a specific assay
will be highly dependent upon the specific concentration range and
solution conditions and is verified as part of system suitability
and calibration for this particular assay within the automated
qualification system.
[0070] Refractive index detectors are bulk property detectors,
i.e., they respond to differences in the refractive index of the
mobile phase when the analyte is eluted from the column.
Consequently, it is necessary to stabilize the refractive index of
the mobile phase in order to achieve good signal to noise ratios
when small analyte peaks are eluted. Testing of the refractive
index detector is, of necessity, a system level test and cannot be
performed at a unit level. The change in refractive index with
concentration is not necessarily a linear phenomenon as illustrated
by the water methanol data shown in FIG. 5. Consequently, it is
necessary within an automated system, to exercise care in the
selection of both the analyte used to probe the linearity of
detector response as well as the solvent used as the mobile phase.
Ideally, a small molecule which is readily available in high purity
and has a wide range of concentrations over which the change in
refractive index is linear should be used for PQ testing of RI
based HPLC systems. Representative data for the variation of
refractive index of sucrose water solutions is shown in FIG. 6.
[0071] The protocols used to qualify systems with absorbance
detectors exploit the high sensitivity of absorbance detection as
well as its linear dynamic range. The mass loading of the columns
in typical reverse phase separations is typically a small fraction
of the column's linear dynamic range. Consequently, by increasing
the concentrations of the reverse phase solutes by a factor of 100
.times. the chromatograms obtained with differential RI (DRI)
detection will possess appropriate signal to noise ratios.
[0072] The detector linearity, injector linearity, injector area
precision, retention time precision and flow rate accuracy with DRI
is performed using pre-mixed mobile phase. The pre-mixed mobile
phase allows the stable baselines required for unambiguous
integration.
[0073] The performance qualification of a chromatograph which uses
an absorbance detector is an assessment of baseline performance
which the majority of chromatographic analyses will require.
Because absorbance detectors have a wide linear dynamic range, the
system performance parameters to be determined are isocratic
retention time precision, peak area precision, and system
linearity. A simple reverse phase separation (C18 column with
methanol:water mobile phase) of stable analytes which cover a range
of moderate values of k' is utilized in the present invention. A
mixture of uracil and caffeine is used to generate chromatograms
with a void marker (uracil) and a well-resolved retained peak
suitable for quantitation. The mobile phase can be pre-mixed or
mixed on-line if a gradient system is used. For gradient systems,
on-line mixing should be used. Table 4 lists the appropriate
control values for a general HPLC system.
3TABLE 4 Control Values Retention Area Time Precision Height
Precision Peak RSD(%) RSD(%) RSD(%) Linearity(R.sup.2) Uracil 1.5
1.5 1.5 N/A Caffeine 1.0 1.0 1.0 >0.999
[0074] These values represent the maximum %RSD values that an
analytical HPLC should generate on a simple isocratic
separation.
[0075] The performance qualification of a fluorescence detector in
the present invention is accomplished according to the following
method.
[0076] Because fluorescence detectors do not have a wide linear
dynamic range, the system performance parameters to be determined
are isocratic retention time precision and peak area precision. A
simple reverse phase separation (C18 column with acetonitrile:water
mobile phase) of a stable analytes with a moderate k' is used.
Anthracene is a native fluorophore which is readily separated on a
C18 reversed phase column and is not strongly sensitive to oxygen
quenching or dimer formation. The mobile phase can be pre-mixed or
mixed on-line in a gradient system. As shown in Table 5 below the
appropriate control values for a general HPLC system.
4TABLE 5 Control Values. Area Retention Time Precision Peak RSD(%)
RSD(%) Height Precision RSD(%) Anthracene 1.0 1.0 1.0
[0077] These values represent the maximum %RSD values that an
analytical HPLC should generate on a simple isocratic separation of
anthracene using a fluorescence detector.
[0078] The performance qualification of refractive index detector
within a chromatography system is automated by utilizing the
principles set forth below using premixed mobile phases.
[0079] Measurement of flow rate accuracy and injection volume
accuracy in the manual qualification of systems is generally based
on measuring the time required to fill a volumetric flask (flow
rate accuracy) or by weighing the mass removed from the sample vial
(injection accuracy). Both techniques are manual and, it is
desirable, in the illustrative embodiment to conduct an analysis
that is subject to automation and less prone to human error.
[0080] The void volume of a chromatographic column is that portion
of the column's nominal volume which is occupied by mobile phase,
it includes the inter-particle and intra-particle volume. It is
usually measured by adding an unretained small molecule, such as
acetone, uracil, sodium nitrate, etc., to the sample mixture and
measuring the product of the flow rate and apparent "retention
time." Equation 3 relates the void volume to column parameters and
flow rate.
V.sub.0=.pi.*d.sub.c.sup.2*.epsilon.*L/4=t.sub.0*V.sub..function.
Equation 3:
[0081] Where d.sub.c=column diameter (cm), .epsilon.=column
porosity, L=column length (cm), t.sub.o="retention time" for
unretained component (min.), and V.sub..function.=volumetric flow
rate (mL/min.)
[0082] Equation 3 can be re-arranged to give the following . .
.
(1/t.sub.o)=(1/V.sub.0)*V.sub.71=1/V.sub.0*(V.sub.f+error) Equation
4:
[0083] Consequently, a plot of the quantity 1/t.sub.o vs.
V.sub..function. will be linear. If it is regressed against a
linear equation of the form, Y=A.sub.o+A.sub.1*X, and the
X-intercept is computed. It will have the form.
X-intercept=-A.sub.o/A=flow rate error Equation 5:
[0084] Referring to FIG. 7, Equation 5 is illustrated. The
calculation has the advantage of computing a volumetric flow rate
error term which includes contributions over the working range of
the solvent delivery system and does not include contributions from
the compositional errors of the pump because the peak is
unretained.
[0085] To determine the linearity of the solvent delivery system
109, in the illustrative embodiment an appropriate sample of a
non-retained compound (such as uracil or sodium nitrate) is eluted
at several flow rates which span the active flow rate range. The
unretained component is eluted at the void volume of the column.
The peak area is related to the injection volume by equation set
forth below:
Area-constant*amount-constant*V.sub.inj*concentration
[0086] When a series of injections are made in which the V.sub.inj
is varied and the sample concentration is held constant, the
equation becomes:
Area-constant*(V.sub.inj+.epsilon.)-constant*V.sub.inj+constant*.epsilon.
[0087] Where .epsilon. is the volumetric error. Once again, if a
plot of peak area is regressed vs. V.sub.inj the X-intercept is
given by.
X-intercept=-constant*.epsilon./constant=-.epsilon.
[0088] FIG. 8 illustrates the above approach
[0089] The x-intercept is an estimate of the volumetric error of
the quantity delivered and includes contributions over the fall
dynamic range of the instrument and estimates the systematic error
in the quantity delivered to the column by the sample manager 110.
This makes the approach appropriate to sample managers in which the
sample is contained within the sample needle as well as those which
transfer the sample to a sample loop.
[0090] In both the measurement of flow rate accuracy and injection
volume accuracy, the values of the x-intercepts establish an error
budget which can then be applied to the assay requirements. For
example, the 0.050 mL/min. error is a 5% error at 1.0 mL/min, but
is only 2% at 2 mL/min. The 0.050 .mu.L error contributes a 2 parts
per thousand error at 25 .mu.L and 1% at 5 .mu.L. This error budget
should then be a part of establishing the system suitability
criteria for a specific assay.
[0091] The qualification of the gradient proportioning system 111
is the measurement of the compositional accuracy based on shifts in
retention time with small changes in composition; the system noise
will be greater, but retention times are not strongly impacted by
baseline noise if the signal to noise ratio is large. The
compositional accuracy measures the degree to which the solvent
management system can generate a specific solvent mixture. To
determine the compositional accuracy, inject the same volumes of
samples while using different combinations of reservoirs in the
chromatography system. The relative standard deviation (%RSD) of
the retention times is measured and those of caffeine should be
fairly consistent. The qualification standard requires the relative
standard deviation to be less than or equal to 2%.
[0092] The qualification of the delay volume 112 of the solvent
management system is the volume of mobile phase that separates the
gradient forming device from the column inlet. Ideally, the delay
volume should be scaled to the void volume of the chromatographic
column to ensure method transferability and efficiency. In prior
manual qualification systems, the delay volume of a gradient
solvent delivery system was measured by the volume required for a
step change in mobile phase composition using a suitable marker
compound (such as acetone or propyl paraben) to arrive at the inlet
connection of the column. The column needed to be removed in such
measurements. Both threshold-based and first derivative-based
calculations have been used to determine the arrival time.
[0093] The delay volume of a gradient chromatography system is the
volume contained in the solvent delivery system from the point at
which the gradient is formed (the gradient proportioning valve in a
low pressure gradient system) or from the first mixing "tee" where
the solvents are combined (high pressure gradient system) to the
column's inlet. Its significance arises from the need to qualify
the gradient proportioning system 111 to ensure that that the delay
volume is delivered to the column before the gradient change in
mobile phase composition begins. It is a temporal offset in the
gradient chromatogram. If all of the components of the sample are
strongly retained in the initial conditions of the gradient, it is
simply overhead that increases the separation time and the time
required to re-equilibrate the column prior to the next injection.
If sample components are not strongly retained at the initial
conditions, the separation will contain peaks which are eluted
under isocratic, gradient and mixed chromatographic modes. The
relationship between k' and peak migration volume is given by:
Fraction of Column Volume Traveled=V.sub.R/{V.sub.0(1+k')}
[0094] When V.sub.R is small, the fraction of the column that a
strongly retained peak travels is very small and the solute is said
to be "focused" at the inlet of the column. When k'=0, the
unretained solute sees the delay volume from the injector to the
column inlet, which should be minimized to reduce sample
dispersion. If the mobile phase is stepped from the initial
conditions to 100% organic modifier, the "focused" solute now
becomes unretained and its migration volume includes the delay
volume as well as the column void volume and the volume of mobile
phase pumped at the initial conditions. Referring to FIG. 9 the
above principle is illustrated.
[0095] In this illustrative embodiment FIG. 9, a mixture of an
unretained and strongly retained compounds (such as uracil and
octanophenone) are injected and the mobile phase composition is
changed in a single step from the initial conditions to 100%
organic modifier. The unretained compound will have a retention
volume equal to the void volume of the column plus the tubing which
connects the column to the injector and detector. The strongly
retained compound will initially be focused at the column inlet and
subsequently eluted as an unretained component after the step to
100% organic modifier. The retention volume of the strongly
retained compound will be the sum of the delay volume, the volume
of the connecting tubing, the void volume of the column, and the
volume of solvent pumped at the initial composition. By subtracting
the void volume (first peak) and the volume at initial conditions
from the retention volume of the strongly retained solute and
correcting for the volume of solvent pumped at the initial
composition, the system delay volume is measured.
[0096] The qualification of the column heater 113 is essential in
those systems that contain column heaters. The qualification of a
column heater 113 is important in order to insure that the column
heater can consistently provide a set temperature because retention
times and mobile phase viscosity are both strongly dependent upon
temperature. The performance of column heaters in a manual
qualification system is determined by placing calibrated
thermocouples or thermistors at, or near, the control point of the
column heater and measuring the difference between the set
temperature and the observed temperature. This approach requires
the use of external meters to read the output of the thermocouple
and is highly subjective because it depends upon the accurate
location of the thermocouple at the control point of the column
heater. Additionally, while this approach confirms that the column
heater is controlling the set point, it does not measure the
effectiveness of the column heater at actually heating the column.
The approach set forth below has the advantage of requiring no
additional devices, no manual data entry, and it is not dependent
upon the location of the temperature probe for its accuracy. When
the column heater is qualified 113, in the illustrative embodiment,
all qualification steps will be performed at a temperature which is
about 10.degree. C. (degrees Celsius) greater than ambient
laboratory temperature. The average retention time measured for the
injections used for the measurement of the sample manager's
precision are a good estimate of the retention time at this base
temperature. The column heater is subsequently set to a temperature
which is 20.degree. C. greater than ambient. After the column is
equilibrated at this new higher temperature a set of injections are
made using the same sample and conditions for the determination of
sample manager precision. The decrease in retention time is
dependant upon the nature of the probe compound the stationary
phase and the mobile phase. A decrease of .about.0.18 minutes has
been empirically observed for the conditions chosen in this
procedure. It is based on the "normal/intended" use of the column
heater within the chromatograph
[0097] The effect of column temperature on retention times is given
by the following:
V.sub.R=V.sub.0*(1+k') Equation 6:
[0098] where V.sub.0=the column's void volume and k'=the capacity
factor for the analyte.
[0099] The capacity factor is related to the equilibrium constant
for the partitioning of the analyte between the stationary phase
and mobile phase by Equation 7.
k'=K*.theta.=C.sub.s*.theta./C.sub.M Equation 7:
[0100] where K=equilibrium constant=C.sub.S/C.sub.M and
.theta.=phase ratio=V.sub.S/V.sub.0 with V.sub.S being the volume
of stationary phase contained within the column and V.sub.0 being
the volume of mobile phase contained within the column. The
equilibrium constant has the usual thermodynamic dependence upon
temperature as given by Equation 8.
ln(K)=lnk'-ln.theta.=-1/RT*.DELTA.G.sup.0 Equation 8:
[0101] where .DELTA.G.sup.0 is the Gibbs free energy associated
with the partitioning of the analyte between the stationary and
mobile phases. A plot of lnk' vs. 1/T (.degree. K.) is linear with
a slope of (-.DELTA.G.sup.0/R) and an intercept of ln.theta.. An
example of two sets of retention time measurements as a function of
column temperature (from 30.degree. C. to 60.degree. C.) is shown
in FIG. 10.
[0102] While equations 6 and 8 predict a non-linear dependence of
retention time with certain temperatures, FIG. 11 demonstrates that
there is a reasonable degree of linearity over a short range of
temperature
[0103] As a result, a simple approach that is amenable to
automation, is the verification of column heaters by measuring the
shift in retention time for a well-equilibrated system with a
column temperature set at 35.degree. C. which is then reset to
45.degree. C. and equilibrated to the new value. This approach as
illustrated in FIG. 11.
[0104] Although the automated test protocol described in the
illustrative embodiment herein is a series of methods that pertain
to the installation, operational and performance qualification of
chromatography systems it should be appreciated that qualification
procedures could be implemented such as to qualify the individual
modules of a chromatography system in the event repairs must be
done, or the like. Similarly, rather than the need for any operator
intervention, qualification could be effected by making the
preparation of the chromatography system completely automated.
[0105] The foregoing has been a description of an illustrative
embodiment of the present invention. The present invention is not
to be limited in scope by the illustrative embodiments described
which are intended as specific illustrations of individual aspects
of the invention, and functionally equivalent methods and
components are within the scope of the invention. Indeed, various
modifications of the invention, in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are intended to
fall within the scope of the appended claims
* * * * *