The importance of software validation increases since the need for high
usability and suitability of software applications grows. In order to reduce
costs and manage risk factors, more and more recommendations and rules have
been established. In the field of pharmacy the vendors of so-called
chromatography data systems (CDSs) had to implement the guidelines of
the Code of Federal Regulations Title 21 (CFR 21) during the last few years
in order to fulfill the increasing requirements. The CFR 21 part 11 deals
with electronic records and signatures. This part is binding for each company
in the regulated environment that wishes to create, edit and sign electronic
information instead of printing them on paper. Subsection CFR 21 part
11.10(h) explains how to perform an input check for manual user entries as
well as for data that will be collected from an external device. In this
article we present an approach performing the double entry method on data
provided by the hardware instrument in order to investigate possible
influences on the raw data by the handling CDS.
A software tool has been written which allows us to communicate with a
high-performance liquid chromatography (HPLC) detector and acquire data from
it. The communication is completely independent of a CDS which is started
separately and connected to the same system. Using this configuration we made
a parallel data acquisition of two instances at the same time possible. Two
CDSs have been tested and for at least one of them it has been shown that a
comparison of the acquired data can be done as with the double entry method
for the data verification. For the second CDS we checked whether it would be
applicable after a few modifications. The given approach could be either used
for a live data verification of produced raw data or as a single test during
a software operational qualification to verify the data acquisition
functionality of the software.
Introduction
High usability and suitability, risk management and data integrity are terms
more and more users of software applications have to consider when they
integrate them into their standard operating procedures. Especially in the
fields of medicine, pharmacy and the food industry any occurrence of risk to
patients and customers leads to the usage of software applications whose
developers established the principles of software validation in their
software development life cycle. Following these guidelines helps to reduce
long-term costs, failure rates and recalls .
Manufacturers and vendors of analytical instruments like high-performance
liquid chromatography (HPLC) or gas chromatography (GC) systems are requested
to implement several parts of the Code of Federal Regulations Title 21 (CRF
21) if they would like to create a validated chromatography data system (CDS)
software package for their product. The CFR 21 deals with the environment of
food and drugs HPLC and GC instruments are mainly used
for.
CFR 21 part 11.10(h)
The Code of Federal Regulations Title 21 (CFR 21) is divided into many parts,
where part 11 deals with electronic records and signatures. The rules within
this part are mandatory for each company that wishes to create, edit and sign
any digital records instead of printing them on paper. Any company that works
with HPLC/GC systems and establishes CFR 21 part 11 requires developers of
CDSs to guarantee the defined guidelines within their software product,
especially subparts 11.10 and 11.30. These ones explain how to control
electronic records in closed and open systems using procedures like input
checks, encryption, signatures and audit trails.
For this article we focused on point (h) of subpart 11.10: Use of device (e.g., terminal) checks to determine, as appropriate, the validity of the source of data input or operational instruction.
That means manually entered as well as automatically incoming data given to
the software application (e.g., CDS) have to be validated and verified. The
validation of data ensures sensible and reasonable inputs. Manual entries of
data or automatically received data can be validated by checks for length,
format and range . The data verification on
the other hand ensures that the incoming data match
the original one. For manual entries there are two methods for the data
verification . The data could either be
entered twice by two separate persons and compared afterwards using the
double entry method or the entered data could be proofread using the original
data as a reference. Both methods are time-consuming, especially when data
are collected manually. The comparison of received data with a reference
could be handled relatively quickly using an algorithm executed by a
computer. But what about data provided by an analytical instrument like a
HPLC system? Usually there are no reference data inside the instrument
available for proofreading and the data will be acquired once by the
connected controller, the CDS software package installed on the computer. To
fulfill CFR 21 part 11.10(h) for data collected from an external instrument,
an interpretation of this part says that a suitable connection between the
hardware system and the controller and an identity verification of the source
data is required . One suitable connection type is based
on the TCP/IP protocol which uses handshakes and checksums in order to
guarantee reliable transport of the data. But for the user themselves it is
not apparent whether there happen to be any faults when preparing the data
for the transport within the device or whether the acquiring software
processes the raw data before making them available for export. We show how
to perform the double entry method for the provided data of a HPLC system
manufactured by Agilent Technologies based on parallel data acquisition using
two instances that communicate with the instrument at the same time.
Methods and materials
Usually one HPLC system will be controlled by one CDS. After an established
connection a commercial CDS usually locks the instrument for a second one in
order to avoid any manipulations to the experiment settings during a sequence
run. But without a second CDS it is not possible to acquire signal data of a
HPLC detector simultaneously within the same run because a second instance is
required to acquire data from one source twice.
Dealing with this problem, new software written in C# has been developed
called Second Controller Instance which is able to connect to a HPLC
system manufactured by Agilent Technologies. In contrast to a fully
functional CDS, our tool searches for a detector module in the HPLC cluster
and uses as little access as possible in order to acquire its signal data
only. The necessary communication is based on the freely available
LICOP library provided by Agilent Technologies
. This library establishes a TCP/IP
connection to the HPLC detector and provides several channels dealing with
the module like sending instructions, monitoring, or acquiring raw data.
After a successful connection the tool uses the two instructions
without quotation marks and send them via an instruction channel in
order to subscribe to the raw data from the given source number. The source
number depends on the given type of HPLC detector and the desired signal.
Additionally these commands involve a module firmware “B.x” and higher
. All experiments in this article
are done with a diode array detector and the desired signal is absorbance
signal no. 1 of the detector. Therefore source number 0 has been chosen here.
After the subscription to the data the so-called RAWD channel defined in the
LICOP library will handle incoming data and provide it for the tool.
This way it is possible to connect to the HPLC instrument even after the lock
of the parallel-running CDS. A LAN connection to the HPLC system which allows
two instances is necessary only. This can be handled by one LAN card that
allows two instances or by two LAN cards in two different HPLC modules.
Analytical instruments
The setup shown in Fig. using the Second Controller Instance has been executed on two different HPLC systems manufactured by
Agilent Technologies. In the further course of this article they will be
called systems A and B. System A consists of a G4225A degasser, G1312B binary
pump, G1367E wellplate autosampler, G1330B autosampler thermostat, G1316C
column compartment and G4212B diode array detector. The modules of system B
are a G1322A degasser, G1311A quaternary pump, G1329A standard autosampler,
G1316A column compartment and G1315D diode array detector.
All experiment sequences shown in Fig. using a network protocol analyzer have been done with HPLC system B.
Chemicals
All experiments with the Second Controller Instance have been done
using an isocratic test sample containing the four substances dimethyl
phthalate, diethyl phthalate, biphenyl and o-terphenyl. These components were
solved in methanol. The mobile phase consisted of a mix of 35 % vol
HPLC-grade water and 65 % vol Acetonitrile and the stationary phase was
an installed Zorbax xDB-C8 column supplied by Agilent Technologies according
to a reverse-phase chromatography configuration. The column had a length of
50 mm, a diameter of 4.6 mm and a pore size of 1.8 µm.
For the network tracking experiments a simpler configuration was used. The
sample was 50 µg mL-1 caffeine
solved in HPLC-grade water. The mobile phase was HPLC-grade water. Due to one
single substance in the sample no separation was needed, and so a restriction
capillary was installed instead of a separation column.
The whole configuration using the Second Controller Instance for a parallel data acquisition. It runs concurrently with the
commercial chromatography data system (CDS) which parameterizes the HPLC
system and starts/stops all experiment sequences.
Experiment setup
Two setups have been created. The first one was used to evaluate the
reliability of a second instance acquiring data in parallel for the double
entry method. The second setup included a network tracking to examine
possible processing of the raw data provided by the device during the storage
procedure.
The network tracking of the data transmitted using TCP over the
ethernet bypasses the not well-known storage procedure of the acquired raw
data of the commercial CDS.
Second Controller Instance
The complete HPLC systems A and B were parameterized and controlled by
commercial CDS OpenLab ChemStation®
(Rev. C.01.07 Build 27) developed and published by Agilent Technologies or by
Chromeleon® (Rev. 6.80 SR15 Build
4656) developed and published by Thermo Fischer without any influence of the
Second Controller Instance. But during the run all generated data
were received in parallel by both the CDS and the Second Controller Instance as shown in Fig. .
Both CDSs were used to set up the following specifications for the
experiments: 1 mL min-1 flow, 1 µL injection volume,
40 ∘C column temperature and 254 nm detection wavelength for
absorbance signal no. 1. These parameters were fixed for each experiment, but
several available detector sampling rates were used by changing the
“expected narrowest peak width at half height” parameter of the HPLC
detector. For HPLC detectors manufactured by Agilent Technologies this
parameter implies a specific combination of sampling rate and signal
filtration. For example, a configured “expected narrowest peak width at half
height” of 0.0125 min (0.75 s) using a G4212B DAD detector leads to a
sampling rate of 20 Hz and a response time of 0.2 s as the filtration
value.
Every experiment condition has been repeated 10 times. After the setup of the
HPLC cluster by the CDS, the Second Controller Instance has been
executed and connected to the same system. Then the Second Controller Instance was requested to subscribe to the detector signal data. The
incoming data handled by the raw data channel (RAWD) of the LICOP
library were interpreted by the
Second Controller Instance tool relating to the data specification
. That way the Second Controller Instance created separate text files for each run containing a
header and the raw data.
Raw data check by network tracking
For the tracking of the data transmitted over the ethernet using TCP the
Wireshark network protocol analyzer tool (v.2.0.5) developed by
Wireshark-Community was used. This tool allows us to catch data
packages between the HPLC device and computer that will be sent and received
via TCP and UDP. As shown in Fig. this constellation
bypasses the unknown storage process of the CDS or LICOP library
used by our Second Controller Instance.
Using this setup a simple run was performed injecting 10 µL of the
caffeine solution. The flow of the pump was set to 1 mL min-1 and the
detector wavelength to 273 nm for absorbance signal no. 1. The temperature
control of the column compartment was turned off here. The provided data were
acquired then either by our Second Controller Instance or CDS
Chromeleon® and caught by Wireshark
at the same moment.
Results and discussion
All experiments using the commercial CDS and Second Controller Instance resulted in chromatograms as shown in Fig.
or similar ones. The four substances of the isocratic test sample have been
separated and correspond to the four peaks. The shown chromatograms in
Fig. were acquired by OpenLab ChemStation® and generated by HPLC system A
using a sampling rate of 20/2.5 Hz and a response time of 0.5/2 s as a
signal filtration parameter. This is the usual result of a CDS before the
data processing is executed including peak detection and peak integration in
order to evaluate the chromatogram.
Chromatogram of dimethyl phthalate (a), diethyl phthalate (b),
biphenyl (c) and o-terphenyl (d) using a sampling
rate of 20 Hz and a response time (signal filtration) of 0.5 s or 2.5 Hz
and a response time of 2 s.
Double entry method
For the data verification of the chromatogram a complete comparison of all
data points between the two instances that acquired the absorbance signal is
necessary as for the double entry method. Such a comparison is shown in
Fig. . The deviation of the signal given
as the difference between the signal value of CDS OpenLab ChemStation® and the Second Controller Instance is plotted against time like in the default chromatogram
(Fig. ). Two experiments are shown using two different
sampling rates and response times. Both plots present an increasing deviation
at the time range of the peaks. Only for the first peak does the deviation
exceed a value of 10-5 or -10-5 mAU. This also applies to the
second peak using a 20 Hz sampling rate and 0.5 s response time.
A closer examination of the data points shows that the deviation completely
depends on the data accuracy given as the available number of decimal places.
The exported data from OpenLab ChemStation® have a single precision which
represents up to 7 digits where the acquired data of the Second Controller Instance have a double precision with 15 digits. So all the
deviations are caused by rounding. Rounding the Second Controller Instance data like for example 150.307349860668 mAU at time point 46 s to
single precision 150.3073 mAU leads to totally equal data points. This
behavior applies for all experiments done with systems A and B and every used
sampling rate/response time.
The phenomenon that the deviation of the second peak in
Fig. got a greater maximum is based on
the sampling rate. A lower sampling rate can lead to a lower peak height
that is visible in
Fig. . That is why the signal value of the second peak
exceeds a value of 100 mAU (single precision with a maximum of four decimal
places now), leading to a deviation greater than 10-5 mAU for a 20 Hz
sampling rate but not for a 2.5 Hz sampling rate.
Signal deviation as the difference between OpenLab ChemStation® and Second Controller Instance when using sampling rates of 20 Hz (0.5 s response time) and
2.5 Hz (2 s response time).
A comparison of the raw data acquired by
Chromeleon® and Second Controller Instance from HPLC system A resulted in
Fig. . In contrast to
Fig. the deviation is much higher and
depends on the configured sampling rate/response time. Additionally there are
two peaks (one negative and one positive) describing one peak in the default
chromatogram. These peaks visualize that the biggest deviation occurs during
the rising and falling areas of the peaks in the chromatogram. That means the
negative deviation increases to a local minimum and decreases afterwards
until the apex of the peak when crossing the abscissa in the deviation plot.
Then the deviation increases once more in a positive way when the peak is
falling. The minimum and maximum seem to be the inflection points caused by
the greatest slope at these points.
This information indicates that there is a time delay between the signal
acquired by Chromeleon® and
Second Controller Instance because this explains why a low change in
the signal leads to a marginal deviation where a great signal slope (e.g., at
the inflection points) induces a high deviation between two data points. As
is visible in Fig. , a higher sampling
rate reduces the maximal deviation due to a smaller time delay between two
data points. In this case the global maximum decreases 5.7-fold when using
20 Hz instead of 2.5 Hz. The dependency on the sampling rate also applies
to HPLC system B.
Signal deviation as the difference between
Chromeleon® and Second Controller Instance when using a sampling rate of 20 Hz (0.5 s response
time) and 2.5 Hz (2 s response time).
Data handling of the LICOP library
The relative large deviations between the stored and exported data of CDS
Chromeleon® and our Second Controller Instance brought us to a more detailed comparison of the raw data
that will be provided by the HPLC device and stored by the corresponding
software package. First of all we wanted to find out whether the two tested
CDSs are using different drivers communicating with the device. But by means
of the Wireshark network protocol analyzer it has been determined
that both CDSs OpenLab ChemStation®
and Chromeleon® are based on the
LICOP library, too. On the one hand the library file exists in the
installation location of both CDSs and on the other hand the commands that
have been sent to the device and caught in the network are equal or similar
to the Second Controller Instance, which is definitely based on the
library.
On the basis of the fact that the LICOP is the only external library
used by the Second Controller Instance whose source code is unknown,
the influence of that library on the incoming data was checked. Therefore, a
single injection of a caffeine solution was performed, resulting in a
chromatogram containing one peak. The data were acquired and stored by the
Second Controller Instance and caught in parallel by the network
analyzer. The provided data of the HPLC device within the TCP packages are
given as hexadecimal values. For the interpretation of the data format a
description file was used . That way
the data which belong to the run of the experiment have been extracted and
converted from hexadecimal to decimal format. These values have the unit
count. In order to compare the raw data, the counts have been converted to
mAU using a factor of 2 097 152 counts per AU, which could be requested
from the HPLC detector. So the conversion from counts to mAU can be done
using Eq. ().
A value-by-value comparison of the stored and caught raw data showed that
both of them are totally equal. The conversion and comparison of all data
points around the peak are presented in the Supplement. That means the
underlying LICOP library does not modify the incoming raw data just
before they are available for the Second Controller Instance.
Processing of the raw data during the storage procedure within CDS Chromeleon®
As with the LICOP library an experiment using the network analyzer
has been performed in order to bypass the data storage procedure of the CDS
(Fig. ). The experiment conditions are similar. The
caffeine solution has been injected, generating a chromatogram with one peak,
and Chromeleon® acquired the data.
Meanwhile, the network was tracked simultaneously.
For the purpose of avoiding incorrect settings several options available from
Chromeleon® have been tried out in
order to get the totally equal raw data that the device provides. In addition
to the usual setup of the device the CDS allows us to configure the handling
of the incoming data. Two parameters called step and
average are available for this. The step parameter can be
set to “auto” or to a fixed value meaning the step width between two data
points in seconds. The “auto” option induces an algorithm which calculates
a dynamic sampling rate and stores the data using it. In order to get
equidistant data as provided by the device we used a fixed step
value. The average parameter defines which kind of reducing method
will be performed if the slice width of the provided data by the device is
lower than the given step value. If the average parameter
is set to off, only every nth data point will be stored. If it is set to
on, the data will be bunched by computing the average of several data points
to form a new one. That means if the given slice width for example is 0.01 s
(100 Hz sampling rate) and the entered step value is 0.2 s (20 Hz
sampling rate), either every fifth data point will be stored only
(average= off) or five data points will be averaged to form one
data point (average= on).
Comparison plots between
Chromeleon® and the caught data
packages of the network generated by HPLC system B. (a) Raw data of
20 Hz without data averaging, (b) raw data of 2.5 Hz without data
averaging, (c, d) time shift of the network data to align the peak
apexes, and (d) deviation as the difference between the
chromatograms in (c, d) computing
Chromeleon® data-network data.
Based on the given HPLC detector of system B, we configured a peak width of
> 0.1 min. That way the detector used an internal sampling rate
of 2.5 Hz to digitize the signal. The step parameter in
Chromeleon® was set to 0.4 s, which
corresponds to the 2.5 Hz sampling rate of the incoming data. For a second
data rate of 20 Hz a peak width of > 0.01 min and a step of
0.05 s were configured. The experiments have been repeated for an activated
and deactivated average parameter.
The results for 20 and 2.5 Hz sampling rates and deactivated averaging are
visible in Fig. a and b. They show the exported
chromatograms of Chromeleon® and
chromatograms formed by the caught data from the network. Both plots have an
apparent time shift between the signals. The exact time shift between the
peak apexes in both chromatograms is 0.4 s. But, in contrast to
Fig. a, in Fig. b
the network data have a delay related to the data of
Chromeleon®. Additionally counting
the number of data shows that the network analyzer caught more data points
than the CDS stored.
In order to check whether the time shift is the only difference between the
signals, the network data have been shifted by +0.4 s in
Fig. a and -0.4 s in
Fig. b to align the retention times. That forms
the chromatograms shown in Fig. c and d. After
the normalizing of the shift there are still apparent deviations in the
rising and falling areas of the peaks. That is why a deviation plot has been
formed in Fig. e, as with the results of the
Second Controller Instance. It is obvious that there is still a
deviation after the time shift, but unlike before it does not depend on the
set sampling rate as much as for the experiment in
Fig. . The wider range of the deviation
plot when using 2.5 Hz is due to a peak broadening when using a more intense
signal filtration . The 2.5 Hz setup uses 2 s
as its response time compared to 0.5 s for the 20 Hz setup. The response
time itself is defined by the American Society for Testing and Materials
(ASTM) as the time required for the signal to rise from 10 % to 90 %
in response to an upward step function . The filter
algorithm used in the Agilent Technologies detector seems to be based on a
moving average filter that includes a Gaussian weighting function
.
The resulting chromatograms using an activated average option are
shown in the Supplement. Even if the provided sampling rate of the device of
20 or 2.5 Hz and the acquiring one defined by the step parameter of
0.05 and 0.4 s, respectively, were intentionally set to the same value to
avoid reducing the number of data points by the CDS, the maximal occurred
deviation has increased here from nearly 17 mAU
(Fig. e) to 44 mAU.
The network tracking shows that any deviations between
Chromeleon® and the Second Controller Instance determined during the double entry experiments are
completely based on unknown, internal processing of
Chromeleon® on the incoming data
before storing them. Even if the provided sampling rate and the desired one
defined in the step option are equal, the data will be modified and
reduced and the pure raw data transmitted over the ethernet are not available
anymore. That means there is an algorithm like an interpolation applied to
the raw data that can not be deactivated. Even if the user has a look into
the detailed manual of the detector in order to determine the sampling rate
of the device that belongs to the configured “narrowest peak width at half
height” parameter and no reduction of data points is needed here, the data
will be modified, and for an activated average option the deviations
are significant here. Furthermore, it is possible to enter a step
value corresponding to a sampling rate that is higher than the provided one.
So non-sampled but estimated data will be stored as raw data.
Conclusions
Using the new written Second Controller Instance has shown that it
is possible to acquire data generated by an HPLC detector manufactured by
Agilent Technologies twice during one and the same run. We found out that the
chromatography data systems (CDSs) OpenLab ChemStation® and
Chromeleon® are based on the
LICOP library for the instrument communication, as with our
Second Controller Instance. The parallel data acquisitions have
shown that the data received by OpenLab ChemStation® are totally equal, except for
the rounding process when it exports its data. That way it was possible to
prove that when two different controllers (OpenLab ChemStation® and Second Controller Instance) subscribe to the same source, we get the same results. So not only
the transport itself via TCP could be ensured, but also the identification of
the source data that will be loaded from the internal device storage and
packaged for the transport.
In practice the second instance could be used here to perform the double
entry approach for the data verification as part of the input check defined
in CFR 21 part 11.10(h). So it should be possible to verify incoming data of
every run in a whole experiment sequence by comparing both data sets before
processing them. Alternatively, the data verification by the double entry
method could be performed once within a software operational qualification as
a test of the receiving data functionality right after the installation of
the software application. Additionally, it is conceivable to use the double
entry generally to compare a new software package with already existing and
validated ones, as done here for the Second Controller Instance.
Regarding CDS Chromeleon®, the
signal deviations which occurred even if the CDS is also based on the
LICOP library are caused by internal data processing during the
storage routine. So it has been shown that the original provided data of the
HPLC device are not accessible when using this software package.
Additionally, without these internal computations the data between the CDSs
and the Second Controller Instance would be totally equal. This fact
shows that a complete comparison of raw data generated by one given HPLC
system under identical conditions always results into two different data sets
if the two tested CDSs are used. So the main problem is that
Chromeleon® only stores the
processed data and not the pure raw data which are gone that way.
In addition it is to highlight how important the setting of the step
parameter in Chromeleon® is because
we focused on setting the corresponding step values to fit the incoming data.
But by the ability to compute more data points than generated by the device
it is possible to store a chromatogram that is influenced by a low sampling
rate (e.g., lower peak height ) but that looks
like a high-rate sampled one. Doing so the integration of the resulting peaks
can lead to significant differences between for example a chromatogram
sampled with 80 Hz acquired by OpenLab ChemStation® and a chromatogram sampled with
20 Hz acquired by Chromeleon® and
stored with 80 Hz.
For the double entry method the advantages of a Second Controller Instance can not be used for
Chromeleon® directly here, but the
approach could be applied if the CDS would compare the data straight before
the storage. This problem and further ones have to be solved when
implementing a second instance for another HPLC manufacturer or even for GC
instruments. For now the only known prerequisite is a second instance allowed
on the LAN connection.
Data availability
The underlying measurement data are not publicly available and can be requested from the authors if required.
The supplement related to this article is available online at: https://doi.org/10.5194/jsss-8-207-2019-supplement.
Author contributions
DTM and DW prepared and performed the data acquisition experiments. DTM implemented the Seconds Controller Instance
and evaluated results. DW, HP and RP advised, reviewed and recommended the corrections of the article.
Competing interests
The authors declare that they have no conflict of interest.
Special issue statement
This article is part of the special issue “Sensors and Measurement Systems 2018”.
It is a result of the “Sensoren und Messsysteme 2018, 19. ITG-/GMA-Fachtagung”, Nürnberg, Germany, from 26 June 2018 to 27 June
2018.
Acknowledgements
We thank AnaTox GmbH and Co. KG for supporting this research by providing all
software packages, required materials, chemicals and HPLC systems.
Review statement
This paper was edited by Andreas Schütze and reviewed by two anonymous referees.
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