Experiments were conducted to assess the effects of small but deliberate changes in
operational factors, such as reducing the length of the capillary between the cigarette valve and the ion volume,
installing a new ferrule, and the day and time of day of measurement. In total, 30 Kentucky 3R4F cigarettes were smoked (10 per day for 3 days) with changes
to the capillary and ferrule made each morning and afternoon of each day in a controlled
manner (Additional Table S1). Overall, the mean ± SD (range) yields per cigarette (n=30) ranged from 27.3 ± 3.3 (18.5–32.5) g/cig for 1,3-butadiene to 387.4 ± 54.2 (293.0–508.0) g/cig for acetaldehyde (Table 2).
By ANOVA, no statistically significant differences (P≥0.05) were found for capillary, ferrule or time of day (a.m. vs p.m.) for any of
the seven constituents (Table 2 in the Supplemental Files). In addition, “treatment”, defined as a combination of
the small changes (e.g., a measurement with a new capillary and ferrule performed
on day 1 in the morning), did not lead to significant differences in the data. Thus,
changing the capillary, ferrule or time of day when measurements are performed does
not affect yield measurements for the LM2X-TOFMS. However, a significant difference
(P<0.05) was seen in day-to-day variation for four of the seven constituents (acetone,
isoprene, benzene and toluene). As a result, further measurements to analyse the day-to-day
variation were carried out.
A further 30 repeat runs were carried out over 5 days with a different number of runs
per day (3, 6, 4, 5 and 12) to specifically analyse the day-to-day variation. In this
test, each 3R4F cigarette sample was removed individually from the conditioning room
immediately before analysis. One-way ANOVA of the 30 measurements showed that day
was not a significant factor for any of the seven analytes (acetaldehyde, P=0.063; 1,3-butadiene, P=0.603; acetone, P=0.510; isoprene, P=0.576; 2-butanone, P=0.639; benzene, P=0.597; toluene, P=0.169).
The raw data (reported as g/puff derived from the instrument algorithm, post toluene calibration) from the repeat
measurements (n=30) were analysed in Minitab to produce control charts for each analyte
to determine whether the LM2X-TOFMS operates in a controlled and stable manner. Apart
from toluene, all data points on the individual charts lay within the control limits
(data not shown). For toluene, one point of the moving range chart was just outside
the upper control limit (UCL). The other 11 measurements on that day showed similar
yields and group around the calculated mean, suggesting that the first point was an
outlier. In the control chart of overall variability across the 5 days (Figure 2), all data points were within the control limits. As shown in Figure 2, there was a gradual shift in mean because the last nine points
were below the mean line. This deviation was noted during data analysis; if observed
during operation, it would trigger further investigation as per the rule set for Shewhart
control charts .
Taken together, the individual control charts for all analytes confirm that, although
there is day-to-day variation, some of which might be due to cigarette variation (typically
4%–10%; ), the overall analytical process of the LM2X-TOFMS shows good stability and control.
The linearity of the LM2X-TOFMS was tested by analysing gas mixtures with certified
concentrations of the seven analytes. During this analysis, the temperature used in
the ideal gas law equation by the internal algorithm was amended from the heated gas
valve temperature (150oC) to room temperature (22`C) as the puff volume (35 mL) was sampled at room temperature. The mean values of the measured response (n=120 puffs per mixture) are presented in
Table 3 (see Supplemental Files).
To establish linearity, the mean values were plotted against the calculated response
for each analyte, a linear fit was chosen, and the R2 values were calculated for each analyte. As an example, Figure 3 shows that the response for 1,3-butadiene was highly linear (R2=0.9922).
The response for acetaldehyde, acetone, 2-butanone, benzene, isoprene and toluene
was also highly linear with R2 values of 0.9999, 0.9999, 0.9995, 0.9996, 1.000 and 0.9999, respectively (Additional Figure S1). Thus, all seven analytes demonstrated excellent linearity across all gas concentrations
Accuracy was evaluated in terms of the relative error, which was determined for the
gas bag measurements (Table 4 in the Supplemental Files). The errors for acetaldehyde, acetone and isoprene were consistent across the minimum,
maximum and mean values. These errors are therefore likely to be systematic and could
be modified by applying a correction factor to the raw data. Systematic errors were
also observed for 2-butanone and benzene, but because the values were small (<10%),
there would be no need to correct the raw data. Non-systematic errors were observed
for 1,3-butadiene and toluene, where the biggest variation occurred at higher concentrations.
However, the error for toluene was small (<10%).
Repeatability and reproducibility
Repeatability (r) is the maximum difference expected between two sample measurements
within a run, whereas reproducibility (R) is the maximum difference between two samples
measured either in different laboratories by different operators or simply by different
operators. Because this was the first commercial LM2X-TOFMS instrument, it was not
possible to measure R in the former way; the present data were also obtained by one
operator. Thus, reproducibility in this study indicates the maximum difference observed
between two measurements, performed on different days at different times (morning
or afternoon). The repeatability and reproducibility of the gas bag measurements are
presented in Table 5 (see Supplemental Files).
As expected, R was larger than r for all analytes at all six gas concentrations except
for one concentration of acetaldehyde (499.5 ppm; Table 5). As a general principle of process control, a coefficient of variation (CV; or relative
standard deviation, RSD) of less than 10% would be considered acceptable ; however, the mean value should also be considered because the CV may be high at
very low concentrations and low at very high concentrations owing to the Horwitz trumpet
effect . Indeed, the biggest variations were observed for lower gas concentrations.
The smallest variation in repeatability (r) was observed for isoprene, for which all
six gas concentrations demonstrated a CV of less than 6%. The second smallest variation
was observed for toluene: for which the CV was less than 9% except at the lowest concentration
(15.21 ppm) which had a CV of 12.1%. The largest variation was observed for acetaldehyde,
which increased from 8.0% for the highest concentration (2000 ppm) to 26.6% for the
lowest concentration (199.5 ppm).
The data provide limits for future reference. For example, in the case of two isoprene
measurements performed on the same day at a yield of 66.7 g/puff, the repeatability should be within 3.0% or 2.0 g/puff. If the measurements were performed on different days (reproducibility), then
the difference should be within 13.4% or 8.9 g/puff.
Repeatability, r, was also assessed on an inter-day (between days) and intra-day (within
day) basis (Table 6 in the Supplemental Files). Day 1 data were used for intra-day results as this was
the first day that the gas bags were used (no sample carry over). Data from all 3
days were used to calculate the inter-day CV.
The stability of the system towards each analyte was further assessed on a per-puff
basis by constructing individual moving range control charts. For a system to be deemed
stable, the points in the charts should lie within the upper (UCL) and lower (LCL)
control limits. This range should also reflect fitness for measurement. Using toluene
as an example (Figure 4), 119 of the 120 data points were within the control limits for both the individual
measurements and moving range charts. Only one of the individual measurements lay
just outside the UCL (Figure 4a, top). Because up to 1 point in 25 can be outside these limits (Shewhart’s criterion
), the analytical process for toluene is considered stable and in control. When the
variability in repeat measurements within a single analytical run was considered (Figure 4b), three points in the moving range chart (bottom) were just outside the UCL; however,
these data indicate the difference between two individual measurements that were within
the UCL (top).
Regarding the other analytes, only 2 of the 29 control charts had data lying outside
Shewhart’s criterion for statistical control: one for isoprene measurements of the
594.6 ppm gas concentration; and one for 1,3-butadiene measurements of the 39.85 ppm
gas concentration. For isoprene, 8 of the 120 data points were outside the control
limits; however, the data displayed a random order, indicating there was no pattern
to these outliers (data not shown). Similarly, for 1,3-butadiene, 8 of the 120 data
points were outside the LCL and UCL. In this instance, however, a cluster of data
points outside the LCL is apparent (Figure 5). These 8 data points were obtained on the first analytical run of day 2 measurements.
The 1,3-butadiene yield decreased during run five; however, this was observed only
during data analysis, so there was no opportunity to investigate; if noted at the
time of measurement, it would trigger further investigation and rejection of the data
set. The other runs made on day 2 (runs 2, 3 and 4) were all within the control limits.
Figure 5a also shows that there was a downward trend in values over the first day and morning of the second
day of measurement, but the data stabilised for the subsequent measurements.
Repeatability of cigarette sample measurements
To further check the repeatability of the system, three different cigarette products
with varying tar yields were analysed for each of the seven vapour-phase analytes.
The mean yield per cigarette (n=30) was determined by smoking each product to the
butt mark. As would be expected, the highest tar yield product, CM6 (NFDPM 14 mg/cig)
produced the highest yield per cigarette for all seven analytes, followed by 3R4F
(NFDPM 9.4 mg/cig) and the commercial cigarette DW (NFDPM 1.9 mg/cig) (Table 7 in the Supplemental Files).
The measurements for the three cigarette products were analysed for repeatability
(r). The RSD was calculated from the average yield of each analyte per product given
in Table 5. Both analyte and product variation were analysed. Regarding product variation, 3R4F
showed the lowest average RSD across the seven analytes at 7.0%, followed by CM6 at
7.1% and the commercial cigarette (DW) at 13.5%. For 3R4F and CM6, all RSD values
were less than the statistically relevant limit of 10%  (i.e., ≤9.7% and ≤9.3%, respectively). By contrast, all RSD values were above 10%
(but ≤16.2%) for DW. This may be because the yields of the DW data were 4–6 times lower than those of the other products, with a proportionally greater impact
In terms of analyte variation, acetone and 2-butanone had the lowest RSD at 8.2%,
followed by benzene (8.8%), isoprene (9.6%), toluene (9.7%), acetaldehyde (10.0%)
and 1,3-butadiene (10.1%) (Table 8 in the Supplemental Files). By coupling a single-channel smoke machine with PI-TOF-MS via a constant flow orifice, Pang et al.  recently carried out an on-line analysis of the same seven compounds in mainstream
smoke from 3R4F reference cigarettes, reporting RSDs below 15% for all analytes, similar to the current values.
Puff-by-puff analysis of cigarette data
The data from the LM2X-TOFMS can also be represented as yield per 35-mL puff, in keeping
with the ISO smoking conditions used throughout this study. Each cigarette was smoked
to the butt mark according to ISO standards (tipping paper length plus 3 mm), resulting
in analyte data for up to 8–10 puffs per cigarette. Each puff was therefore compared
with its counterpart in other runs. For example, all the puff-one data were averaged
to obtain the mean ± SD yield for puff one (Figure 6). Because some runs had a slightly different puff number, all graphs were normalised
to the minimum consistent puff number. The number of cigarettes analysed per puff
number are given in the legend.
Although the yields vary per puff, trends are apparent for most of the analytes. Apart
from 2-butanone, all analytes had a visibly higher yield in the first puff than in
the second puff. After the second puff, the yield increased with increasing puff number.
For all three cigarette products, the first puff had the highest yield of 1,3-butadiene,
isoprene and benzene. Similar puff-by-puff behaviour of analytes has been observed
in previous studies [16,20].
With increasing puff number from puff 3 to the final puff, there was an increase in
mean concentration for all seven analytes for DW and 3R4F. For CM6, there was an overall
increase in mean concentration with increasing puff number from puff 3, but six of
the seven analytes, acetaldehyde, acetone, 2-butanone, benzene, isoprene and toluene,
demonstrated a slightly lower mean for puff 5 as compared with puff 4.
For 1,3-butadiene, isoprene and benzene yields in CM6 products, puff one was unique
to any other puff in the run. For CM6 products, acetaldehyde, 2-butanone and toluene exhibited the highest yield
in their final puff. For 3R4F products, acetaldehyde, acetone, 2-butanone and toluene
exhibited the highest yield in their final puff. For the commercial DW cigarette, only toluene exhibited the highest yield in its final
puff. Notably, the large variation (i.e., SD) in the first puff indicates how different
the lighting puff can be from cigarette to cigarette. This has been noted in previous
studies , and is thought to be due to the increase in temperature in the tobacco, from room
temperature to approximately 900°C.
Operational range of the LM2X-TOFMS and data comparison
From the certified gas mixture measurements in Table 3, a working operational range for the LM2X-TOFMS was determined. The operational range
was also corrected for accuracy, as defined by the relative error reported in Table 4. The operational range and corrected operational range are summarized in Table 9 (in the Supplemental Files).
The accuracy correction factors were also applied to the cigarette yield data (Table 10 in the Supplemental Files). The average (ISO) yield ± SD are the yields directly calculated
by the LM2X-TOFMS, whereas the corrected yield ± SD are the yields that have been
calculated based on the accuracy.
The corrected LM2X-TOFMS yield data were compared with internal and external published
cigarette yield data. First, carbonyl measurements from the LM2X-TOFMS for 3R4F and
CM6 were compared with published data generated by the CORESTA-recommended method
for measuring carbonyls, involving smoke collection in impinger traps, derivatisation with 2,4-dinitrophenylhydrazine,
separation of carbonyl hydrazones by reversed-phase HPLC and detection by ultra violet
or diode array  (Figure 7a). The 3R4F reference data, measured by the LM2X-TOFMS and corrected by accuracy (see
Table 10), were then compared with comparison data generated internally by BAT (mean values
per cig from 50 runs), collected by different offline methods (Figure 7b).
Overall, the data sets compare well (Table 11 in the Supplemental Files). Notably, the standard deviations of the measurements performed
on the LM2X-TOFMS seem to be smaller than those of the CORESTA data set . The online PI-TOFMS analysis of 3R4F mainstream smoke by Pang et al.  also reported similar values.